Materials and methods for treatment of alpha-1 antitrypsin deficiency

ABSTRACT

The present application provides materials and methods for treating a patient with Alpha-1 antitrypsin deficiency (AATD) both ex vivo and in vivo. In addition, the present application provides materials and methods for editing the SERPINA1 gene in a cell by genome editing.

RELATED APPLICATIONS

This application is a national phase filing under 35 USC § 371 ofInternational PCT Application No. PCT/IB2016/001845, filed Dec. 1, 2016,which claims the benefit and priority of U.S. Provisional ApplicationNo. 62/261,661, filed Dec. 1, 2015; and U.S. Provisional Application No.62/324,056, filed Apr. 18, 2016; the contents of each of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present application provides materials and methods for treating apatient with alpha-1 antitrypsin deficiency (AATD) both ex vivo and invivo. In addition, the present application provides materials andmethods for modulating the expression, function, and/or activity of theSERPINA1 gene and/or the alpha-1 antitrypsin (AAT) protein in a cell bygenome editing.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form(filename: CRIS-010_NO1US_SequenceListing_ST25.txt; 13,386 KB in size;created May 29, 2018), which is incorporated herein by reference in itsentirety and forms part of the disclosure.

BACKGROUND OF THE INVENTION

Alpha-1 antitrypsin deficiency (AATD) is an autosomal recessive disordercharacterized by an increased risk for chronic obstructive pulmonarydisease, including, emphysema, airflow obstruction, and/or chronicbronchitis, with evident manifestation in adults by the 4^(th)-5^(th)decades; liver disease, in a small portion of affected patients, withmanifestation as obstructive jaundice; and increased aminotransferaselevels in the early period of life. The liver disease in adults can evenmanifest as fibrosis and cirrhosis in the absence of a history of livercomplications during childhood. Patients with AATD also have a higherincidence of hepatocellular carcinoma. (See e.g., Stoller J K,Aboussouan L S, “A review of alpha1-antitrypsin deficiency.” Am J RespirCrit Care Med 2012, 185(3):246-259; Teckman J H, “Liver disease inalpha-1 antitrypsin deficiency: current understanding and futuretherapy.” COPD 2013, 10 Suppl 1:35-43; and Stoller J K, et al, “Alpha-1Antitrypsin Deficiency.” In: GeneReviews®. Edited by Pagon R A, et al.Seattle (Wash.); 1993).

The diagnosis of AATD is confirmed by demonstration of a lowconcentration of alpha-1 antitrypsin (AAT) in serum, followed bydetection of a functionally deficient AAT protein or detection ofbi-allelic pathogenic variants in SERPINA1, the gene encoding alpha-1antitrypsin. (See e.g., Aboussouan L S, Stoller J K; “Detection ofalpha-1 antitrypsin deficiency: a review.” Respir Med 2009,103(3):335-341). The prevalence of AATD is about 40:100K (Swedishancestry-50-60:100K). More than 100 variants of the AAT gene (SERPINA1)have been described, but the majority of the patients (at least with theliver phenotype) are homozygous for PI Z (PI, GLU342LYS ON MIA[dbSNP:rs28929474]).

Current therapy for AATD includes: standard therapy for chronicobstructive pulmonary disease, which includes bronchodilators, inhaledcorticosteroids, pulmonary rehabilitation, supplemental oxygen, andvaccinations (e.g., influenza and pneumococcal). Specific therapy forAATD, including periodic intravenous infusion of pooled human serumalpha-antitrypsin (augmentation therapy) is also used. (See e.g.,Mohanka M, Khemasuwan D, Stoller J K, “A review of augmentation therapyfor alpha-1 antitrypsin deficiency.” Expert Opin Biol Ther 2012,12(6):685-700). Individuals with end-stage lung disease often have toundergo lung transplantation. A major treatment for patients with theliver disease is liver transplantation because other approaches, such asconventional therapies (chemical chaperones, stimulation of autophagy,antifibrotic therapy), often have a minimal effect.

Some newer approaches have been tested recently. (See e.g., Wewers M D,et al., “Replacement therapy for alpha 1-antitrypsin deficiencyassociated with emphysema.” N Engl J Med 1987, 316(17):1055-1062; WewersM D, Crystal R G, “Alpha-1 antitrypsin augmentation therapy.” COPD 2013,10 Suppl 1:64-67; and Teckman J H, “Lack of effect of oral4-phenylbutyrate on serum alpha-1-antitrypsin in patients withalpha-1-antitrypsin deficiency: a preliminary study.” J PediatrGastroenterol Nutr 2004, 39(1):34-37). ARC-AAT is a single dose Phase 1clinical study and ALNY-AAT is a Phase 1 clinical study. Both studiesare harnessing the RNA interference approach to reduce the amount ofmutant AAT deposited in hepatocytes. However, these approaches do notaddress the lack of normal AAT in the circulation, and depend uponcombination with augmentation therapy. In addition, rAAV1-CB-hAAT is aPhase 2 clinical study that combines downregulation of endogenous AATwith microRNA and simultaneous upregulation of exogenous AAT. (See e.g.,Flotte T R, et al., “Phase 2 clinical trial of a recombinantadeno-associated viral vector expressing alpha1-antitrypsin: interimresults.” Hum Gene Ther 2011, 22(10):1239-1247; Mueller C, et al.,“Sustained miRNA-mediated knockdown of mutant AAT with simultaneousaugmentation of wild-type AAT has minimal effect on global liver miRNAprofiles.” Mol Ther 2012, 20(3):590-600).

Genome engineering refers to the strategies and techniques for thetargeted, specific modification of the genetic information (genome) ofliving organisms. Genome engineering is a very active field of researchbecause of the wide range of possible applications, particularly in theareas of human health; the correction of a gene carrying a harmfulmutation, for example, or to explore the function of a gene. Earlytechnologies developed to insert a gene into a living cell, such astransgenesis, were often limited by the random nature of the insertionof the new sequence into the genome. The new gene was usually positionedblindly, and may have inactivated or disturbed the functioning of othergenes, or even caused severe unwanted effects. Furthermore, thesetechnologies generally offered no degree of reproducibility, as therewas no guarantee that the new sequence would be inserted at the sameplace in two different cells. More recent genome engineering strategies,such as ZFNs, TALENs, HEs and MegaTALs, enable a specific area of theDNA to be modified, thereby increasing the precision of the correctionor insertion compared to early technologies, and offering some degree ofreproducibility. Despite this, such recent genome engineering strategieshave limitations.

Despite efforts from researchers and medical professionals worldwide whohave been trying to address AATD, and despite the promise of genomeengineering approaches, there still remains a critical need fordeveloping safe and effective treatments for AATD.

Prior approaches addressing AATD have severe limitations, such aslimited success and lack of adequate available therapies. The presentinvention solves these problems by using genome engineering tools tocreate permanent changes to the genome that can restore the AAT proteinactivity with a single treatment. Thus, the present invention correctsthe underlying genetic defect causing the disease.

SUMMARY OF THE INVENTION

Provided herein are cellular, ex vivo and in vivo methods for creatingpermanent changes to the genome by inserting, deleting, correcting, ormodulating the expression or function of one or more mutations or exonswithin or near the SERPINA1 gene or other DNA sequences that encoderegulatory elements of the SERPINA1 gene or knocking-in SERPINA1 cDNA orminigene into a safe harbor locus by genome editing and restoringalpha-1-antitrypsin (AAT) protein activity, which can be used to treatalpha-1 antitrypsin deficiency (AATD). Also provided herein arecomponents, kits and compositions for performing such methods. Alsoprovided are cells produced by them.

Provided herein is a method for editing a SERPINA1 gene in a human cellby genome editing, the method comprising the step of introducing intothe human cell one or more deoxyribonucleic acid (DNA) endonucleases toeffect one or more single-strand breaks (SSBs) or one or moredouble-strand breaks (DSBs) within or near the SERPINA1 gene or otherDNA sequences that encode regulatory elements of the SERPINA1 gene thatresults in at least one of a permanent insertion, deletion, correction,or modulation of expression or function of one or more mutations orexons within or near or affecting the expression or function of theSERPINA1 gene, or within or near a safe harbor locus that results in apermanent insertion of the SERPINA1 gene or minigene, and results inrestoration of alpha-1-antitrypsin (AAT) protein activity.

Also provided herein is a method for inserting a SERPINA1 gene in ahuman cell by genome editing, the method comprising the step of:introducing into the human cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near a safe harbor locus thatresults in a permanent insertion of the SERPINA1 gene or minigene, andresults in restoration of alpha-1-antitrypsin (AAT) protein activity.

In one aspect, provided herein is a method for editing the SERPINA1 genein a human cell by genome editing comprising introducing into the cellone or more deoxyribonucleic acid (DNA) endonucleases to effect one ormore single-strand breaks (SSBs) or double-strand breaks (DSBs) withinor near the SERPINA1 gene or other DNA sequences that encode regulatoryelements of the SERPINA1 gene of the cell that results in permanentdeletion, insertion, or correction of one or more mutations or exonswithin or near the SERPINA1 gene, or within or near a safe harbor locusthat results in permanent insertion of the SERPINA1 gene or minigene,and restoration of alpha-1-antitrypsin (AAT) protein activity.

In another aspect, provided herein is an ex vivo method for treating apatient with alpha-1 antitrypsin deficiency (AATD) comprising the stepsof: creating a patient specific induced pluripotent stem cell (iPSC);editing within or near the SERPINA1 gene of the iPSC or other DNAsequences that encode regulatory elements of the SERPINA1 gene of theiPSC or editing within or near a safe harbor locus of the iPSC;differentiating the genome edited iPSC into a hepatocyte; and implantingthe hepatocyte into the patient.

In some embodiments, the step of creating a patient specific inducedpluripotent stem cell (iPSC) comprises: isolating a somatic cell fromthe patient; and introducing a set of pluripotency-associated genes intothe somatic cell to induce the cell to become a pluripotent stem cell.In some embodiments, the somatic cell is a fibroblast. In someembodiments, the set of pluripotency-associated genes is one or more ofthe genes selected from the group consisting of OCT4, SOX2, KLF4, Lin28,NANOG and cMYC.

The step of editing within or near a SERPINA1 gene or other DNAsequences that encode regulatory elements of the SERPINA1 gene of theiPSC or editing within or near a safe harbor locus of the SERPINA1 geneof the iPSC or editing within or near a locus of the first exon of theSERPINA1 gene of the iPSC can comprise introducing into the iPSC one ormore deoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the SERPINA1 gene or other DNA sequences that encode regulatoryelements of the SERPINA1 gene that results in a permanent insertion,correction, deletion, or modulation of expression or function of one ormore mutations or exons within or near or affecting the expression orfunction of the SERPINA1 gene or within or near a safe harbor locus thatresults in a permanent insertion of the SERPINA1 gene results inrestoration of AAT protein activity. The safe harbor locus can beselected from the group consisting of: AAVS1 (PPP1R12C), ALB, Angptl3,ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1,TF, and TTR. The safe harbor locus can be selected from the groupconsisting of: exon 1-2 of AAVS1 (PPP1R12C), exon 1-2 of ALB, exon 1-2of Angptl3, exon 1-2 of ApoC3, exon 1-2 of ASGR2, exon 1-2 of CCR5, exon1-2 of FIX (F9), exon 1-2 of G6PC, exon 1-2 of Gys2, exon 1-2 of HGD,exon 1-2 of Lp(a), exon 1-2 of Pcsk9, exon 1-2 of Serpina1, exon 1-2 ofTF, and exon 1-2 of TTR.

In some embodiments, the step of editing the SERPINA1 gene of the iPSCcomprises introducing into the iPSC one or more deoxyribonucleic acid(DNA) endonucleases to effect one or more double-strand breaks (DSBs)within or near the SERPINA1 gene that results in permanent deletion,insertion, or correction of one or more mutations within or near theSERPINA1 gene and restoration of alpha-1-antitrypsin (AAT) proteinactivity.

In some embodiments, the step of differentiating the genome edited iPSCinto a liver progenitor cell or a hepatocyte comprises one or more ofthe following: contacting the genome edited iPSC with one or more ofactivin, B27 supplement, FGF4, HGF, BMP2, BMP4, Oncostatin M, orDexametason.

In some embodiments, the step of implanting the hepatocyte into thepatient comprises implanting the hepatocyte into the patient bytransplantation, local injection, or systemic infusion, or combinationsthereof.

In another aspect, provided herein is an ex vivo method for treating apatient with alpha-1 antitrypsin deficiency (AATD) comprising the stepsof: performing a biopsy of the patients liver; isolating a liverspecific progenitor cell or primary hepatocyte; editing within or nearthe SERPINA1 gene or other DNA sequences that encode regulatory elementsof the SERPINA1 gene of the progenitor cell or primary hepatocyte orediting within or near a safe harbor locus of the progenitor cell orprimary hepatocyte; and implanting the genome-edited progenitor cell orprimary hepatocyte into the patient.

In some embodiments, the step of isolating a liver specific progenitorcell or primary hepatocyte from the patient comprises: perfusion offresh liver tissues with digestion enzymes, cell differentialcentrifugation, and cell culturing, or combinations thereof.

In some embodiments, the step of editing within or near the SERPINA1gene of the progenitor cell or primary hepatocyte or other DNA sequencesthat encode regulatory elements of the SERPINA1 gene of the progenitorcell or primary hepatocyte or editing within or near a safe harbor locusof the white blood cell of the progenitor cell or primary hepatocytecomprises introducing into the progenitor cell or primary hepatocyte oneor more deoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the SERPINA1 gene or other DNA sequences that encode regulatoryelements of the SERPINA1 gene that results in permanent insertion,correction, deletion, or modulation of expression or function of one ormore mutations or exons within or near or affecting the expression orfunction of the SERPINA1 gene or editing within or near a safe harborlocus that results in permanent deletion, insertion, or correction ofone or more mutations within or near the SERPINA1 gene and restorationof alpha-1-antitrypsin (AAT) protein activity. The safe harbor locus canbe selected from the group consisting of: AAVS1 (PPP1R12C), ALB,Angptl3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9,Serpina1, TF, and TTR. The safe harbor locus can be selected from thegroup consisting of: exon 1-2 of AAVS1 (PPP1R12C), exon 1-2 of ALB, exon1-2 of Angptl3, exon 1-2 of ApoC3, exon 1-2 of ASGR2, exon 1-2 of CCR5,exon 1-2 of FIX (F9), exon 1-2 of G6PC, exon 1-2 of Gys2, exon 1-2 ofHGD, exon 1-2 of Lp(a), exon 1-2 of Pcsk9, exon 1-2 of Serpina1, exon1-2 of TF, and exon 1-2 of TTR.

In some embodiments, the step of implanting the progenitor cell orprimary hepatocyte into the patient comprises implanting the progenitorcell or primary hepatocyte into the patient by transplantation, localinjection, or systemic infusion, or combinations thereof.

In an aspect, provided herein is an ex vivo method for treating apatient with alpha-1 antitrypsin deficiency (AATD), the methodcomprising the steps of: i) isolating a mesenchymal stem cell from thepatient; ii) editing within or near a SERPINA1 gene or other DNAsequences that encode regulatory elements of the SERPINA1 gene of themesenchymal stem cell or editing within or near a safe harbor locus ofthe SERPINA1 gene of the mesenchymal cell; iii) differentiating thegenome-edited mesenchymal stem cell into a hepatocyte; and iv)implanting the hepatocyte into the patient.

In an aspect, provided herein is an ex vivo method for treating apatient with alpha-1 antitrypsin deficiency (AATD) comprising the stepsof: performing a biopsy of the patient's bone marrow; isolating amesenchymal stem cell; editing the SERPINA1 gene of the stem cell;differentiating the stem cell into a hepatocyte; and implanting thehepatocyte into the patient.

The mesenchymal stem cell can be isolated from the patient's bone marrowby performing a biopsy of the patient's bone marrow or the mesenchymalstem cell can be isolated from peripheral blood.

In some embodiments, the step of isolating a mesenchymal stem cellcomprises: aspiration of bone marrow and isolation of mesenchymal cellsby density centrifugation using Percoll™.

In some embodiments, the step of editing within or near the SERPINA1gene of the stem cell or other DNA sequences that encode regulatoryelements of the SERPINA1 gene of the mesenchymal stem cell comprisesintroducing into the mesenchymal stem cell one or more deoxyribonucleicacid (DNA) endonucleases to effect one or more single-strand breaks(SSBs) or double-strand breaks (DSBs) within or near the SERPINA1 geneor other DNA sequences that encode regulatory elements of the SERPINA1gene that results in permanent deletion, insertion, correction, ormodulation of expression or function of one or more mutations or exonswithin or near or affecting the expression or function of the SERPINA1gene or within or near a safe harbor locus that results of one or moremutations within or near the SERPINA1 gene that results in permanentinsertion of the SERPINA1 gene or minigene, and restoration ofalpha-1-antitrypsin (AAT) protein activity. The safe harbor locus can beselected from the group consisting of: AAVS1 (PPP1R12C), ALB, Angptl3,ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1,TF, and TTR. The safe harbor locus can be selected from the groupconsisting of: exon 1-2 of AAVS1 (PPP1R12C), exon 1-2 of ALB, exon 1-2of Angptl3, exon 1-2 of ApoC3, exon 1-2 of ASGR2, exon 1-2 of CCR5, exon1-2 of FIX (F9), exon 1-2 of G6PC, exon 1-2 of Gys2, exon 1-2 of HGD,exon 1-2 of Lp(a), exon 1-2 of Pcsk9, exon 1-2 of Serpina1, exon 1-2 ofTF, and exon 1-2 of TTR.

In some embodiments, the step of differentiating the genome-editedmesenchymal stem cell into a hepatocyte comprises one or more of thefollowing to differentiate the genome edited mesenchymal stem cell intoa hepatocyte: contacting the genome edited mesenchymal stem cell withone or more of insulin, transferrin, FGF4, HGF, or bile acids.

In some embodiments, the step of implanting the hepatocyte into thepatient comprises implanting the hepatocyte into the patient bytransplantation, local injection, or systemic infusion, or combinationsthereof.

In another aspect, provided herein is an in vivo method for treating apatient with alpha-1 antitrypsin deficiency (AATD) comprising the stepof editing the SERPINA1 gene in a cell of the patient, or other DNAsequences that encode regulatory elements of the SERPINA1 gene, orediting within or near a safe harbor locus in a cell of the patient.

In some embodiments, the step of editing the SERPINA1 gene in a cell ofthe patient comprises introducing into the cell one or moredeoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the SERPINA1 gene or other DNA sequences that encode regulatoryelements of the SERPINA1 gene that results in permanent deletion,insertion, or correction or modulation of one or more mutations or exonswithin the SERPINA1 gene that results in permanent insertion of theSERPINA1 gene or minigene, and restoration of AAT protein activity.

In some embodiments, the step of editing the SERPINA1 gene in a cell ofthe patient comprises introducing into the cell one or moredeoxyribonucleic acid (DNA) endonucleases to effect one or moredouble-strand breaks (DSBs) within or near the SERPINA1 gene or DNAsequences that encode regulatory elements of the SERPINA1 gene orediting within or near a safe harbor locus of the SERPINA1 gene thatresults in permanent deletion, insertion, correction, or correction, ormodulation of expression or function of one or more exons within or nearor affecting the expression or function of the SERPINA1 gene andrestoration of alpha-1-antitrypsin (AAT) protein activity.

In some embodiments, the one or more DNA endonucleases is a Cas1, Cas1B,Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 andCsx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2,Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2,Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2,Csf3, Csf4, or Cpf1 endonuclease; a homolog thereof, recombination ofthe naturally occurring molecule, codon-optimized, or modified versionthereof, and combinations of any of the foregoing.

In some embodiments, the method comprises introducing into the cell oneor more polynucleotides encoding the one or more DNA endonucleases. Insome embodiments, the method comprises introducing into the cell one ormore ribonucleic acids (RNAs) encoding the one or more DNAendonucleases. In some embodiments, the one or more polynucleotides orone or more RNAs is one or more modified polynucleotides or one or moremodified RNAs. In some embodiments, the method comprises introducinginto the cell one or more DNA endonucleases wherein the endonuclease isa protein or polypeptide.

In some embodiments, the method further comprises introducing into thecell one or more guide ribonucleic acids (gRNAs). In some embodiments,the one or more gRNAs are single-molecule guide RNA (sgRNAs). In someembodiments, the one or more gRNAs or one or more sgRNAs is one or moremodified gRNAs, one or more modified sgRNAs, or combinations thereof.

In some embodiments, the one or more DNA endonucleases is pre-complexedwith one or more gRNAs or one or more sgRNAs, or combinations thereof.

In some embodiments, the method further comprises introducing into thecell a polynucleotide donor template comprising a part of the wild-typeSERPINA1 gene or minigene (comprised of, natural or synthetic enhancerand promoter, one or more exons, and natural or synthetic introns, andnatural or synthetic 3′UTR and polyadenylation signal), DNA sequencesthat encode wild-type regulatory elements of the SERPINA1 gene, and/orcDNA. In some embodiments, the part of the wild-type SERPINA1 gene orcDNA can be exon 1, exon 2, exon 3, exon 4, exon 5, intronic regions,fragments or combinations thereof, or the entire SERPINA1 gene or cDNA.In some embodiments, the donor template is either a single or doublestranded polynucleotide. In some embodiments, the donor template hashomologous arms to the 14q32.13 region.

In some embodiments, the method further comprises introducing into thecell one guide ribonucleic acid (gRNA) and a polynucleotide donortemplate comprising at least a portion of the wild-type SERPINA1 gene.In some embodiments, the method further comprises introducing into thecell one guide ribonucleic acid (gRNA) and a polynucleotide donortemplate comprising at least a portion of a codon optimized or modifiedSERPINA1 gene. The one or more DNA endonucleases is one or more Cas9endonucleases that effect one double-strand break (DSB) at a DSB locuswithin or near the SERPINA1 gene (or codon optimized or modifiedSERPINA1 gene) or other DNA sequences that encode regulatory elements ofthe SERPINA1 gene, or within or near a safe harbor locus thatfacilitates insertion of a new sequence from the polynucleotide donortemplate into the chromosomal DNA at the locus or safe harbor locus thatresults in a permanent insertion or correction of a part of thechromosomal DNA of the SERPINA1 gene or other DNA sequences that encoderegulator elements of the SERPINA1 gene proximal to the locus or safeharbor locus and restoration of AAT protein activity. In someembodiments, the gRNA comprises a spacer sequence that is complementaryto a segment of the locus or safe harbor locus. In some embodiments,proximal means nucleotides both upstream and downstream of the locus orsafe harbor locus.

In some embodiments, the method further comprises introducing into thecell two guide ribonucleic acid (gRNAs) and a polynucleotide donortemplate comprising at least a portion of the wild-type SERPINA1 gene,and wherein the one or more DNA endonucleases is two or more Cas9endonucleases that effect a pair of double-strand breaks (DSBs), thefirst at a 5′ DSB locus and the second at a 3′ DSB locus, within or nearthe SERPINA1 gene or other DNA sequences that encode regulatory elementsof the SERPINA1 gene, or within or near a safe harbor locus thatfacilitates insertion of a new sequence from the polynucleotide donortemplate into the chromosomal DNA between the 5′ DSB locus and the 3′DSB locus that results in permanent insertion or correction of thechromosomal DNA between the 5′ DSB locus and the 3′ DSB locus within ornear the SERPINA1 gene or other DNA sequences that encode regulatoryelements of the SERPINA1 gene, or within or near a safe harbor locus andrestoration of AAT protein activity, and wherein the first guide RNAcomprises a spacer sequence that is complementary to a segment of the 5′DSB locus and the second guide RNA comprises a spacer sequence that iscomplementary to a segment of the 3′ DSB locus.

In some embodiments, the one or more gRNAs are one or moresingle-molecule guide RNA (sgRNAs). In some embodiments, the one or moregRNAs or one or more sgRNAs is one or more modified gRNAs or one or moremodified sgRNAs.

In some embodiments, the one or more DNA endonucleases is pre-complexedwith one or more gRNAs or one or more sgRNAs.

In some embodiments, the part of the wild-type SERPINA1 gene or cDNA isthe entire SERPINA1 gene or cDNA.

In some embodiments, the donor template is either single or doublestranded. In some embodiments, the donor template has homologous arms tothe 14q32.13 region.

In some embodiments, the DSB, or 5′ DSB and 3′ DSB are in the firstexon, first intron, or both the first exon and first intron of theSERPINA1 gene.

In some embodiments, the gRNA or sgRNA is directed to one or more of thefollowing pathological variants: rs764325655, rs121912713, rs28929474,rs17580, rs121912714, rs764220898, rs199422211, rs751235320,rs199422210, rs267606950, rs55819880, rs28931570.

In some embodiments, the insertion or correction is by homology directedrepair (HDR).

In some embodiments, the method further comprises introducing into thecell two guide ribonucleic acid (gRNAs), and wherein the one or more DNAendonucleases is two or more Cas9 endonucleases that effect a pair ofdouble-strand breaks (DSBs), the first at a 5′ DSB locus and the secondat a 3′ DSB locus, within or near the SERPINA1 gene or other DNAsequences that encode regulatory elements of the SERPINA1 gene, orwithin or near a safe harbor locus that causes a deletion of thechromosomal DNA between the 5′ DSB locus and the 3′ DSB locus thatresults in permanent deletion of the chromosomal DNA between the 5′ DSBlocus and the 3′ DSB locus within or near the SERPINA1 gene or other DNAsequences that encode regulatory elements of the SERPINA1 gene, orwithin or near a safe harbor locus and restoration of AAT proteinactivity, and wherein the first guide RNA comprises a spacer sequencethat is complementary to a segment of the 5′ DSB locus and the secondguide RNA comprises a spacer sequence that is complementary to a segmentof the 3′ DSB locus.

In some embodiments, the two gRNAs are two single-molecule guide RNA(sgRNAs). In some embodiments, the two gRNAs or two sgRNAs are twomodified gRNAs or two modified sgRNAs.

In some embodiments, the one or more DNA endonucleases is pre-complexedwith one or two gRNAs or one or two sgRNAs.

In some embodiments, both the 5′ DSB and 3′ DSB are in or near eitherthe first exon, second exon, third exon, fourth exon or fifth exon orintrons of the SERPINA1 gene.

In some embodiments, the correction is by homology directed repair(HDR).

In some embodiments, the deletion is a deletion of 1 kb or less.

In some embodiments, the Cas9 or Cpf1 mRNA, gRNA, and donor template areeither each formulated separately into lipid nanoparticles or allco-formulated into a lipid nanoparticle.

In some embodiments, the Cas9 or Cpf1 mRNA, gRNA, and donor template areformulated into separate exosomes or are co-formulated into an exosome.

In some embodiments, the Cas9 or Cpf1 mRNA is formulated into a lipidnanoparticle, and both the gRNA and donor template are delivered to thecell by a viral vector. In some embodiments, the viral vector is anadeno-associated virus (AAV) vector. In some embodiments, the AAV vectoris an AAV6 vector.

The Cas9 or Cpf1 mRNA can be formulated into a lipid nanoparticle, andthe gRNA can be delivered to the cell by electroporation and donortemplate can be delivered to the cell by a viral vector. In someembodiments, the viral vector is an adeno-associated virus (AAV) vector.In some embodiments, the AAV vector is an AAV6 vector.

In some embodiments, the SERPINA1 gene is located on Chromosome 14:1,493,319-1,507,264 (Genome Reference Consortium—GRCh38/hg38).

The restoration of AAT protein activity can be compared to wild-type ornormal AAT protein activity.

In another aspect, provided herein is one or more guide ribonucleicacids (gRNAs) comprising a spacer sequence selected from the groupconsisting of the nucleic acid sequences in Examples 1-6 and in SEQ IDNOs 54,860-68,297: for editing the SERPINA1 gene in a cell from apatient with alpha-1 antitrypsin deficiency. In some embodiments, theone or more gRNAs are one or more single-molecule guide RNAs (sgRNAs).In some embodiments, the one or more gRNAs or one or more sgRNAs is oneor more modified gRNAs or one or more modified sgRNAs.

In another aspect, provided herein is one or more guide ribonucleicacids (gRNAs) comprising a spacer sequence selected from the groupconsisting of the nucleic acid sequences in SEQ ID NOs: 1-54,859 forediting a safe harbor locus in a cell from a patient with alpha-1antitrypsin deficiency, wherein the safe harbor locus is selected fromthe group consisting of AAVS1 (PPP1R12C), ALB, Angptl3, ApoC3, ASGR2,CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, and TTR. Insome embodiments, the one or more gRNAs are one or more single-moleculeguide RNAs (sgRNAs). In some embodiments, the one or more gRNAs or oneor more sgRNAs is one or more modified gRNAs or one or more modifiedsgRNAs.

In another aspect, provided herein are cells that have been modified bythe preceding methods to permanently correct one or more mutationswithin the SERPINA1 gene and restore AAT protein activity. Furtherprovided herein are methods for ameliorating Alpha-1 AntitrypsinDeficiency (AATD) by the administration of cells that have been modifiedby the preceding methods to an AATD patient.

In some embodiments, the methods and compositions of the disclosurecomprise one or more modified guide ribonucleic acids (gRNAs).Non-limiting examples of modifications can comprise one or morenucleotides modified at the 2′ position of the sugar, in someembodiments a 2′-O-alkyl, 2′-O-alkyl-O-alkyl, or 2′-fluoro-modifiednucleotide. In some embodiments, RNA modifications include 2′-fluoro,2′-amino or 2′ O-methyl modifications on the ribose of pyrimidines,abasic residues, desoxy nucleotides, or an inverted base at the 3′ endof the RNA.

In some embodiments, the one or more modified guide ribonucleic acids(gRNAs) comprise a modification that makes the modified gRNA moreresistant to nuclease digestion than the native oligonucleotide.Non-limiting examples of such modifications include those comprisingmodified backbones, for example, phosphorothioates, phosphorothyos,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages.

Various other aspects are described and exemplified herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of materials and methods for treatment of Alpha-1Antitrypsin Deficiency (AATD) disclosed and described in thisspecification can be better understood by reference to the accompanyingfigures, in which:

FIG. 1A is an illustration of a plasmid (referred to herein as “CTx-1”)comprising a codon optimized gene for S. pyogenes Cas9 endonuclease. TheCTx-1 plasmid also comprises a gRNA scaffold sequence, which includes a20 bp spacer sequence from the sequences listed in Examples 1 and 8.

FIG. 1B is an illustration of a plasmid (referred to herein as “CTx-2”)comprising a different codon optimized gene for S. pyogenes Cas9endonuclease. The CTx-2 plasmid also comprises a gRNA scaffold sequence,which includes a 20 bp spacer sequence from the sequences listed inExamples 1 and 8.

FIG. 1C is an illustration of a plasmid (referred to herein as “CTx-3”)comprising yet another different codon optimized gene for S. pyogenesCas9 endonuclease. The CTx-3 plasmid also comprises a gRNA scaffoldsequence, which includes a 20 bp spacer sequence from the sequenceslisted in Examples 1 and 8.

FIG. 2A is an illustration depicting the type II CRISPR/Cas system.

FIG. 2B is another illustration depicting the type II CRISPR/Cas system.

FIG. 3 is a graph depicting cutting efficiencies of gRNAs transcribed invitro and transfected using messenger Max into HEK293T cells thatconstitutively express Cas9, as evaluated using TIDE analysis.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1-2,032 are 20 bp spacer sequences for targeting an AAVS1(PPP1R12C) gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 2,033-2,203 are 20 bp spacer sequences for targeting anAAVS1 (PPP1R12C) gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 2,204-2,221 are 20 bp spacer sequences for targeting anAAVS1 (PPP1R12C) gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 2,222-2,230 are 20 bp spacer sequences for targeting anAAVS1 (PPP1R12C) gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 2,231-2,305 are 20 bp spacer sequences for targeting anAAVS1 (PPP1R12C) gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 2,306-3,481 are 22 bp spacer sequences for targeting anAAVS1 (PPP1R12C) gene with an Acidominococcus, Lachnospiraceae, andFrancisella novicida Cpf1 endonuclease.

SEQ ID NOs: 3,482-3,649 are 20 bp spacer sequences for targeting an Albgene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 3,650-3,677 are 20 bp spacer sequences for targeting an Albgene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 3,678-3,695 are 20 bp spacer sequences for targeting an Albgene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 3,696-3,700 are 20 bp spacer sequences for targeting an Albgene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 3,701-3,724 are 20 bp spacer sequences for targeting an Albgene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 3,725-4,103 are 22 bp spacer sequences for targeting an Albgene with an Acidominococcus, Lachnospiraceae, and Francisella novicidaCpf1 endonuclease.

SEQ ID NOs: 4,104-4,448 are 20 bp spacer sequences for targeting anAngpt13 gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 4,449-4,484 are 20 bp spacer sequences for targeting anAngpt13 gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 4,485-4,507 are 20 bp spacer sequences for targeting anAngpt13 gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 4,508-4,520 are 20 bp spacer sequences for targeting anAngpt13 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 4,521-4,583 are 20 bp spacer sequences for targeting anAngpt13 gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 4,584-5,431 are 22 bp spacer sequences for targeting anAngpt13 gene with an Acidominococcus, Lachnospiraceae, and Francisellanovicida Cpf1 endonuclease.

SEQ ID NOs: 5,432-5,834 are 20 bp spacer sequences for targeting anApoC3 gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 5,835-5,859 are 20 bp spacer sequences for targeting anApoC3 gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 5,860-5,862 are 20 bp spacer sequences for targeting anApoC3 gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 5,863-5,864 are 20 bp spacer sequences for targeting anApoC3 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 5,865-5,876 are 20 bp spacer sequences for targeting anApoC3 gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 5,877-6,108 are 22 bp spacer sequences for targeting anApoC3 gene with an Acidominococcus, Lachnospiraceae, and Francisellanovicida Cpf1 endonuclease.

SEQ ID NOs: 6,109-7,876 are 20 bp spacer sequences for targeting anASGR2 gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 7,877-8,082 are 20 bp spacer sequences for targeting anASGR2 gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 8,083-8,106 are 20 bp spacer sequences for targeting anASGR2 gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 8,107-8,118 are 20 bp spacer sequences for targeting anASGR2 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 8,119-8,201 are 20 bp spacer sequences for targeting anASGR2 gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 8,202-9,641 are 22 bp spacer sequences for targeting anASGR2 gene with an Acidominococcus, Lachnospiraceae, and Francisellanovicida Cpf1 endonuclease.

SEQ ID NOs: 9,642-9,844 are 20 bp spacer sequences for targeting a CCR5gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 9,845-9,876 are 20 bp spacer sequences for targeting a CCR5gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 9,877-9,890 are 20 bp spacer sequences for targeting a CCR5gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 9,891-9,892 are 20 bp spacer sequences for targeting a CCR5gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 9,893-9,920 are 20 bp spacer sequences for targeting a CCR5gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 9,921-10,220 are 22 bp spacer sequences for targeting a CCR5gene with an Acidominococcus, Lachnospiraceae, and Francisella novicidaCpf1 endonuclease.

SEQ ID NOs: 10,221-11,686 are 20 bp spacer sequences for targeting an F9gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 11,687-11,849 are 20 bp spacer sequences for targeting an F9gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 11,850-11,910 are 20 bp spacer sequences for targeting an F9gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 11,911-11,935 are 20 bp spacer sequences for targeting an F9gene with a 7T. denticola Cas9 endonuclease.

SEQ ID NOs: 11,936-12,088 are 20 bp spacer sequences for targeting an F9gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 12,089-14,229 are 22 bp spacer sequences for targeting an F9gene with an Acidominococcus, Lachnospiraceae, and Francisella novicidaCpf1 endonuclease.

SEQ ID NOs: 14,230-15,245 are 20 bp spacer sequences for targeting aG6PC gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 15,246-15,362 are 20 bp spacer sequences for targeting aG6PC gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 15,363-15,386 are 20 bp spacer sequences for targeting aG6PC gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 15,387-15,395 are 20 bp spacer sequences for targeting aG6PC gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 15,396-15,485 are 20 bp spacer sequences for targeting aG6PC gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 15,486-16,580 are 22 bp spacer sequences for targeting aG6PC gene with an Acidominococcus, Lachnospiraceae, and Francisellanovicida Cpf1 endonuclease.

SEQ ID NOs: 16,581-22,073 are 20 bp spacer sequences for targeting aGys2 gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 22,074-22,749 are 20 bp spacer sequences for targeting aGys2 gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 22,750-20,327 are 20 bp spacer sequences for targeting aGys2 gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 23,028-23,141 are 20 bp spacer sequences for targeting aGys2 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 23,142-23,821 are 20 bp spacer sequences for targeting aGys2 gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 23,822-32,253 are 22 bp spacer sequences for targeting aGys2 gene with an Acidominococcus, Lachnospiraceae, and Francisellanovicida Cpf1 endonuclease.

SEQ ID NOs: 32,254-33,946 are 20 bp spacer sequences for targeting anHGD gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 33,947-34,160 are 20 bp spacer sequences for targeting anHGD gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 34,161-34,243 are 20 bp spacer sequences for targeting anHGD gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 34,244-34,262 are 20 bp spacer sequences for targeting anHGD gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 34,263-34,463 are 20 bp spacer sequences for targeting anHGD gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 34,464-36,788 are 22 bp spacer sequences for targeting anHGD gene with an Acidominococcus, Lachnospiraceae, and Francisellanovicida Cpf1 endonuclease.

SEQ ID NOs: 36,789-40,583 are 20 bp spacer sequences for targeting anLp(a) gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 40,584-40,993 are 20 bp spacer sequences for targeting anLp(a) gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 40,994-41,129 are 20 bp spacer sequences for targeting anLp(a) gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 41,130-41,164 are 20 bp spacer sequences for targeting anLp(a) gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 41,165-41,532 are 20 bp spacer sequences for targeting anLp(a) gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 41,533-46,153 are 22 bp spacer sequences for targeting anLp(a) gene with an Acidominococcus, Lachnospiraceae, and Francisellanovicida Cpf1 endonuclease.

SEQ ID NOs: 46,154-48,173 are 20 bp spacer sequences for targeting aPCSK9 gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 48,174-48,360 are 20 bp spacer sequences for targeting aPCSK9 gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 48,361-48,396 are 20 bp spacer sequences for targeting aPCSK9 gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 48,397-48,410 are 20 bp spacer sequences for targeting aPCSK9 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 48,411-48,550 are 20 bp spacer sequences for targeting aPCSK9 gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 48,551-50,344 are 22 bp spacer sequences for targeting aPCSK9 gene with an Acidominococcus, Lachnospiraceae, and Francisellanovicida Cpf1 endonuclease.

SEQ ID NOs: 50,345-51,482 are 20 bp spacer sequences for targeting aSerpina1 gene as a safe harbor locus with a S. pyogenes Cas9endonuclease.

SEQ ID NOs: 51,483-51,575 are 20 bp spacer sequences for targeting aSerpina1 gene as a safe harbor locus with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 51,576-51,587 are 20 bp spacer sequences for targeting aSerpina1 gene as a safe harbor locus with a S. thermophilus Cas9endonuclease.

SEQ ID NOs: 51,588-51,590 are 20 bp spacer sequences for targeting aSerpina1 gene as a safe harbor locus with a T. denticola Cas9endonuclease.

SEQ ID NOs: 51,591-51,641 are 20 bp spacer sequences for targeting aSerpina1 gene as a safe harbor locus with a N. meningitides Cas9endonuclease.

SEQ ID NOs: 51,642-52,445 are 22 bp spacer sequences for targeting aSerpina1 gene as a safe harbor locus with an Acidominococcus,Lachnospiraceae, and Francisella novicida Cpf1 endonuclease.

SEQ ID NOs: 52,446-53,277 are 20 bp spacer sequences for targeting a TFgene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 53,278-53,363 are 20 bp spacer sequences for targeting a TFgene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 53,364-53,375 are 20 bp spacer sequences for targeting a TFgene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 53,376-53,382 are 20 bp spacer sequences for targeting a TFgene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 53,383-53,426 are 20 bp spacer sequences for targeting a TFgene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 53,427-54,062 are 22 bp spacer sequences for targeting a TFgene with an Acidominococcus, Lachnospiraceae, and Francisella novicidaCpf1 endonuclease.

SEQ ID NOs: 54,063-54,362 are 20 bp spacer sequences for targeting a TTRgene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 54,363-54,403 are 20 bp spacer sequences for targeting a TTRgene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 54,404-54,420 are 20 bp spacer sequences for targeting a TTRgene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 54,421-54,422 are 20 bp spacer sequences for targeting a TTRgene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 54,423-54,457 are 20 bp spacer sequences for targeting a TTRgene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 54,458-54,859 are 22 bp spacer sequences for targeting a TTRgene with an Acidominococcus, Lachnospiraceae, and Francisella novicidaCpf1 endonuclease.

SEQ ID NOs: 54,860-61,324 are 20 bp spacer sequences for targeting aSERPINA1 gene (other than as a safe harbor locus) with a S. pyogenesCas9 endonuclease.

SEQ ID NOs: 61,325-61,936 are 20 bp spacer sequences for targeting aSERPINA1 gene (other than as a safe harbor locus) with a S. aureus Cas9endonuclease.

SEQ ID NOs: 61,937-62,069 are 20 bp spacer sequences for targeting aSERPINA1 gene (other than as a safe harbor locus) with a S. thermophilusCas9 endonuclease.

SEQ ID NOs: 62,070-62,120 are 20 bp spacer sequences for targeting aSERPINA1 gene (other than as a safe harbor locus) with a T. denticolaCas9 endonuclease.

SEQ ID NOs: 62,121-62,563 are 20 bp spacer sequences for targeting aSERPINA1 gene (other than as a safe harbor locus) with a N. meningitidesCas9 endonuclease.

SEQ ID NOs: 62,564-68,297 are 22 bp spacer sequences for targeting aSERPINA1 gene (other than as a safe harbor locus) with anAcidominococcus, Lachnospiraceae, and Francisella novicida Cpf1endonuclease.

DETAILED DESCRIPTION

Alpha-1 Antitrypsin Deficiency (AATD)

AATD is caused by mutations, or more rarely by deletions, to theSERPINA1 gene. The SERPINA1 gene is located at 14q32.13, with genomiccoordinates (GRCh38) at chr14: 1,493,319-1,507,264. SERPINA1 iscomprised of five exons and has a total length of 12.2 kb. The gene hastwo promoters, with one controlling expression in macrophages. Multipletranscript variants have been reported; however, they all encode thesame AAT protein. About 95% of AATD results from pathological alleles PIZ or PI S or their combination.

The SERPINA1 gene encodes alpha-1-antitrypsin (AAT), also known asprotease inhibitor (PI), a major plasma serine protease inhibitor. AATcomplexes predominantly with elastase, but also with other serineproteases. An important inhibitory action of AAT is against neutrophilelastase, a protease that degrades elastin of the alveolar walls as wellas other structural proteins in a variety of tissues.

Therapeutic Approach

As the known forms of AATD are monogenic disorders with recessiveinheritance, it is likely that correcting one of the mutant alleles percell will be sufficient for correction and restoration or partialrestoration of AATD function. The correction of one allele can coincidewith one copy that remains with the original mutation, or a copy thatwas cleaved and repaired by non-homologous end joining (NHEJ) andtherefore was not properly corrected. Bi-allelic correction can alsooccur. Various editing strategies that can be employed for specificmutations are discussed below.

Correction of one or possibly both of the mutant alleles provides animportant improvement over existing or potential therapies, such asintroduction of SERPINA1 expression cassettes through lentivirusdelivery and integration. Gene editing has the advantage of precisegenome modification and lower adverse effects, for example, the mutationcan be corrected by the insertions or deletions that arise due to theNHEJ repair pathway. If the patient's SERPINA1 gene has an inserted ordeleted base, a targeted cleavage can result in a NHEJ-mediatedinsertion or deletion that restores the frame. Missense mutations canalso be corrected through NHEJ-mediated correction using one or moreguide RNA. The ability or likelihood of the cut(s) to correct themutation can be designed or evaluated based on the local sequence andmicro-homologies. NHEJ can also be used to delete segments of the gene,either directly or by altering splice donor or acceptor sites throughcleavage by one gRNA targeting several locations, or several gRNAs. Thismay be useful if an amino acid, domain or exon contains the mutationsand can be removed or inverted, or if the deletion otherwise restoredfunction to the protein. Pairs of guide strands have been used fordeletions and corrections of inversions.

Alternatively, the donor for correction by HDR contains the correctedsequence with small or large flanking homology arms to allow forannealing. HDR is essentially an error-free mechanism that uses asupplied homologous DNA sequence as a template during DSB repair. Therate of HDR is a function of the distance between the mutation and thecut site so choosing overlapping or nearby target sites is important.Templates can include extra sequences flanked by the homologous regionsor can contain a sequence that differs from the genomic sequence, thusallowing sequence editing.

In addition to correcting mutations by NHEJ or HDR, a range of otheroptions are possible. If there are small or large deletions or multiplemutations, a cDNA can be knocked in that contains the exons affected. Afull length cDNA can be knocked into any “safe harbor”—i.e.,non-deleterious insertion point that is not the SERPINA1 gene itself—,with or without suitable regulatory sequences. If this construct isknocked-in near the SERPINA1 regulatory elements, it should havephysiological control, similar to the normal gene. Two or more (e.g., apair) nucleases can be used to delete mutated gene regions, though adonor would usually have to be provided to restore function. In thiscase two gRNA and one donor sequence would be supplied.

Provided herein are methods to correct the specific mutation in the geneby inducing a double stranded break with Cas9 and a sgRNA or a pair ofdouble stranded breaks around the mutation using two appropriate sgRNAs,and to provide a donor DNA template to induce Homology-Directed Repair(HDR). In some embodiments, the donor DNA template can be a short singlestranded oligonucleotide, a short double stranded oligonucleotide, along single or double stranded DNA molecule. These methods use gRNAs anddonor DNA molecules for each of the variants of SERPINA1.

Provided herein are methods to knock-in SERPINA1 cDNA or a minigene(comprised of one or more exons and introns or natural or syntheticintrons) into the locus of the corresponding gene. These methods use apair of sgRNA targeting the first exon and/or the first intron of theSERPINA1 gene. In some embodiments, the donor DNA is single or doublestranded DNA having homologous arms to the 17q21 region.

Provided herein are methods to knock-in SERPINA1 cDNA or a minigene(comprised of one or more exons and introns or natural or syntheticintrons) into the locus of the hot-spot, e.g., ALB gene. These methodsuse a pair of sgRNA targeting the first exon and/or the first intron ofthe gene located in the liver hotspot. In some embodiments, the donorDNA is single or double stranded DNA having homologous arms to thecorresponding region.

Provided herein are cellular, ex vivo and in vivo methods for usinggenome engineering tools to create permanent changes to the genomeby: 1) correcting, by insertions or deletions that arise due to theimprecise NHEJ pathway, one or more mutations within or near theSERPINA1 gene or other DNA sequences that encode regulatory elements ofthe SERPINA1 gene, 2) correcting, by HDR, one or more mutations withinor near the SERPINA1 gene or other DNA sequences that encode regulatoryelements of the SERPINA1 gene, or 3) deletion of the mutant regionand/or knocking-in SERPINA1 cDNA or minigene (comprised of, natural orsynthetic enhancer and promoter, one or more exons, and natural orsynthetic introns, and natural or synthetic 3′UTR and polyadenylationsignal) into the gene locus or a safe harbor locus of the SERPINA1 gene,and restoring AAT protein activity. Such methods use endonucleases, suchas CRISPR-associated (CRISPR/Cas9, Cpf1, and the like) nucleases, topermanently delete, insert, edit, correct, or replace one or more exonsor portions thereof (i.e., mutations within or near the coding and/orsplicing sequences) or insert in the genomic locus of the SERPINA1 geneor other DNA sequences that encode regulatory elements of the SERPINA1gene. In this way, examples set forth in the present disclosure can helpto restore the reading frame or the wild-type sequence of, or otherwisecorrect, the gene with a single treatment (rather than deliver potentialtherapies for the lifetime of the patient). SERPINA1 gene.

Provided herein are methods for treating a patient with AATD. Anembodiment of such method is an ex vivo cell based therapy. For example,a patient specific induced pluripotent stem cell (iPSC) is created.Then, the chromosomal DNA of these iPS cells is edited using thematerials and methods described herein. Next, the genome-edited iPSCsare differentiated into hepatocytes. Finally, the hepatocytes areimplanted into the patient.

Another embodiment of such method is an ex vivo cell based therapy. Forexample, a biopsy of the patient's liver is performed. Then, a liverspecific progenitor cell or primary hepatocyte is isolated from thepatient, e.g., by a biopsy. Next, the chromosomal DNA of theseprogenitor cells or primary hepatocytes is edited using the materialsand methods described herein. Finally, the genome-edited progenitorcells or primary hepatocytes are implanted into the patient.

Yet another embodiment of such method is an ex vivo cell based therapy.For example, a biopsy of the patients bone marrow is performed. Then, amesenchymal stem cell is isolated from the patient, which can beisolated from the patient's bone marrow, e.g., by biopsy, or peripheralblood. Next, the chromosomal DNA of these mesenchymal stem cells isedited using the materials and methods described herein. Next, thegenome-edited mesenchymal stem cells are differentiated intohepatocytes. Finally, these hepatocytes are implanted into the patient.

One advantage of an ex vivo cell therapy approach is the ability toconduct a comprehensive analysis of the therapeutic prior toadministration. All nuclease based therapeutics have some level ofoff-target effects. Performing gene correction ex vivo allows one tofully characterize the corrected cell population prior to implantation.Aspects of the invention include sequencing the entire genome of thecorrected cells to ensure that the off-target cuts, if any, are ingenomic locations associated with minimal risk to the patient.Furthermore, populations of specific cells, including clonalpopulations, can be isolated prior to implantation.

Another advantage of ex vivo cell therapy relates to genetic correctionin iPSCs compared to other primary cell sources. iPSCs are prolific,making it easy to obtain the large number of cells that will be requiredfor a cell based therapy. Furthermore, iPSCs are an ideal cell type forperforming clonal isolations. This allows screening for the correctgenomic correction, without risking a decrease in viability. Incontrast, other potential cell types, such as primary hepatocytes, areviable for only a few passages and difficult to clonally expand. Thus,manipulation of AATD iPSCs will be much easier, and will shorten theamount of time needed to make the desired genetic correction.

Another embodiment of such method is an in vivo based therapy. In thismethod, the chromosomal DNA of the cells in the patient is correctedusing the materials and methods described herein.

An advantage of in vivo gene therapy is the ease of therapeuticproduction and administration. The same therapeutic approach and therapywill have the potential to be used to treat more than one patient, forexample a number of patients who share the same or similar genotype orallele. In contrast, ex vivo cell therapy typically requires using apatient's own cells, which are isolated, manipulated and returned to thesame patient.

Also provided herein is a cellular method for editing the SERPINA1 genein a cell by genome editing. For example, a cell is isolated from apatient or animal. Then, the chromosomal DNA of the cell is edited usingthe materials and methods described herein.

The methods of the invention, regardless of whether a cellular or exvivo or in vivo method, involves one or a combination of thefollowing: 1) correcting, by insertions or deletions that arise due tothe imprecise NHEJ pathway, one or more mutations within or near theSERPINA1 gene or other DNA sequences that encode regulatory elements ofthe SERPINA1 gene, 2) correcting, by HDR, one or more mutations withinor near the SERPINA1 gene or other DNA sequences that encode regulatoryelements of the SERPINA1 gene, or 3) deletion of the mutant regionand/or knocking in SERPINA1 cDNA or a minigene (comprised of one or moreexons or introns or natural or synthetic introns) or introducingexogenous SERPINA1 DNA or cDNA sequence or a fragment thereof into thelocus of the gene or at a heterologous location in the genome (such as asafe harbor locus, such as, e.g., targeting an AAVS1 (PPP1R12C), an ALBgene, an Angptl3 gene, an ApoC3 gene, an ASGR2 gene, a CCR5 gene, a FIX(F9) gene, a G6PC gene, a Gys2 gene, an HGD gene, a Lp(a) gene, a Pcsk9gene, a Serpina1 gene, a TF gene, and a TTR gene). Assessment ofefficiency of HDR mediated knock-in of cDNA into the first exon canutilize cDNA knock-in into “safe harbor” sites such as: single-strandedor double-stranded DNA having homologous arms to one of the followingregions, for example: ApoC3 (chr11:116829908-116833071), Angptl3(chr1:62,597,487-62,606,305), Serpina1 (chr14:94376747-94390692), Lp(a)(chr6:160531483-160664259), Pcsk9 (chr1:55,039,475-55,064,852), FIX(chrX:139,530,736-139,563,458), ALB (chr4:73,404,254-73,421,411), TTR(chr18:31,591,766-31,599,023), TF (chr3:133,661,997-133,779,005), G6PC(chr17:42,900,796-42,914,432), Gys2 (chr12:21,536,188-21,604,857), AAVS1(PPP1R12C) (chr19:55,090,912-55,117,599), HGD(chr3:120,628,167-120,682,570), CCR5 (chr3:46,370,854-46,376,206), ASGR2(chr17:7,101,322-7,114,310). Both the correction and knock-in strategiesutilize a donor DNA template in Homology-Directed Repair (HDR). HDR ineither strategy may be accomplished by making one or moredouble-stranded breaks (DSBs) at specific sites in the genome by usingone or more endonucleases.

For example, the NHEJ correction strategy can involve restoring thereading frame in the SERPINA1 gene by inducing one single stranded breakor double stranded break in the gene of interest with one or more CRISPRendonucleases and a gRNA (e.g., crRNA+tracrRNA, or sgRNA), or two ormore single stranded breaks or double stranded breaks in the gene ofinterest with two or more CRISPR endonucleases and two or more sgRNAs.This approach can require development and optimization of sgRNAs for theSERPINA1 gene.

For example, the HDR correction strategy involves restoring the readingframe in the SERPINA1 gene by inducing one double stranded break in thegene of interest with one or more CRISPR endonucleases and gRNA (e.g.,crRNA+tracrRNA, or sgRNA), or two or more double stranded breaks in thegene of interest with one or more CRISPR endonucleases and two or moreappropriate sgRNAs, in the presence of a donor DNA template introducedexogenously to direct the cellular DSB response to Homology-DirectedRepair (the donor DNA template can be a short single strandedoligonucleotide, a short double stranded oligonucleotide, a long singleor double stranded DNA molecule). This approach requires development andoptimization of gRNAS and donor DNA molecules for the major variant ofthe SERPINA1 gene (PI Z).

For example, the knock-in strategy involves knocking-in SERPINA1 cDNA ora minigene (comprised of, natural or synthetic enhancer and promoter,one or more exons, and natural or synthetic introns, and natural orsynthetic 3′UTR and polyadenylation signal) into the locus of the geneusing a gRNA (e.g., crRNA _+tracrRNA, or sgRNA) or a pair of sgRNAstargeting upstream of or in the first or other exon and/or intron of theSERPINA1 gene, or in a safe harbor site (such as AAVS1 (PPP1R12C), ALB,Angptl3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9,Serpina1, TF, and/or TTR). The donor DNA will be single or doublestranded DNA having homologous arms to the 14q32.13 region.

For example, the deletion strategy involves deleting one or moremutations in one or more of the five exons of the SERPINA1 gene usingone or more endonucleases and two or more gRNAs or sgRNAs.

The advantages for the above strategies (correction and knock-in anddeletion) are similar, including in principle both short and long termbeneficial clinical and laboratory effects. In addition, it may be thatonly a low percentage of AAT activity is required to provide therapeuticbenefit. Another advantage for all strategies is that most patients havelow-level gene and protein activity, therefore suggesting thatadditional protein expression, for example following gene correction,should not necessarily lead to an immune response against the targetgene product. The knock-in approach does provide one advantage over thecorrection or deletion approach—the ability to treat all patients versusonly a subset of patients. While there are common mutations in thisgene, there are also many other possible mutations, and using theknock-in method could treat all of them. The other issue with geneediting in this manner is the need for a DNA donor for HDR.

In addition to the above genome editing strategies, another strategyinvolves modulating expression, function, or activity of SERPINA1 byediting in the regulatory sequence.

In addition to the editing options listed above, Cas9 or similarproteins can be used to target effector domains to the same target sitesthat can be identified for editing, or additional target sites withinrange of the effector domain. A range of chromatin modifying enzymes,methylases or demethylases can be used to alter expression of the targetgene. One possibility is increasing the expression of the AAT protein ifthe mutation leads to lower activity. These types of epigeneticregulation have some advantages, particularly as they are limited inpossible off-target effects.

A number of types of genomic target sites are present in addition tomutations in the coding and splicing sequences.

The regulation of transcription and translation implicates a number ofdifferent classes of sites that interact with cellular proteins ornucleotides. Often the DNA binding sites of transcription factors orother proteins can be targeted for mutation or deletion to study therole of the site, though they can also be targeted to change geneexpression. Sites can be added through non-homologous end joining (NHEJ)or direct genome editing by homology directed repair (HDR). Increaseduse of genome sequencing, RNA expression and genome-wide studies oftranscription factor binding have increased the ability to identify howthe sites lead to developmental or temporal gene regulation. Thesecontrol systems may be direct or may involve extensive cooperativeregulation that can require the integration of activities from multipleenhancers. Transcription factors typically bind 6-12 bp-long degenerateDNA sequences. The low level of specificity provided by individual sitessuggests that complex interactions and rules are involved in binding andthe functional outcome. Binding sites with less degeneracy may providesimpler means of regulation. Artificial transcription factors can bedesigned to specify longer sequences that have less similar sequences inthe genome and have lower potential for off-target cleavage. Any ofthese types of binding sites can be mutated, deleted or even created toenable changes in gene regulation or expression (Canver, M. C. et al.,Nature (2015)).

Another class of gene regulatory regions having these features ismicroRNA (miRNA) binding sites. miRNAs are non-coding RNAs that play keyroles in post-transcriptional gene regulation. miRNA may regulate theexpression of 30% of all mammalian protein-encoding genes. Specific andpotent gene silencing by double stranded RNA (RNAi) was discovered, plusadditional small noncoding RNA (Canver, M. C. et al., Nature (2015)).The largest class of noncoding RNAs important for gene silencing aremiRNAs. In mammals, miRNAs are first transcribed as a long RNAtranscripts, which can be separate transcriptional units, part ofprotein introns, or other transcripts. The long transcripts are calledprimary miRNA (pri-miRNA) that include imperfectly base-paired hairpinstructures. These pri-miRNA are cleaved into one or more shorterprecursor miRNAs (pre-miRNAs) by Microprocessor, a protein complex inthe nucleus, involving Drosha.

Pre-miRNAs are short stem loops ˜70 nucleotides in length with a2-nucleotide 3′-overhang that are exported, into the mature 19-25nucleotide miRNA:miRNA* duplexes. The miRNA strand with lower basepairing stability (the guide strand) is loaded onto the RNA-inducedsilencing complex (RISC). The passenger guide strand (marked with *),may be functional, but is usually degraded. The mature miRNA tethersRISC to partly complementary sequence motifs in target mRNAspredominantly found within the 3′ untranslated regions (UTRs) andinduces posttranscriptional gene silencing (Bartel, D. P. Cell 136,215-233 (2009); Saj, A. & Lai, E. C. Curr Opin Genet Dev 21, 504-510(2011)).

miRNAs are important in development, differentiation, cell cycle andgrowth control, and in virtually all biological pathways in mammals andother multicellular organisms. miRNAs are also involved in cell cyclecontrol, apoptosis and stem cell differentiation, hematopoiesis,hypoxia, muscle development, neurogenesis, insulin secretion,cholesterol metabolism, aging, viral replication and immune responses.

A single miRNA can target hundreds of different mRNA transcripts, whilean individual transcript can be targeted by many different miRNAs. Morethan 28645 microRNAs have been annotated in the latest release ofmiRBase (v.21). Some miRNAs are encoded by multiple loci, some of whichare expressed from tandemly co-transcribed dusters. The features allowfor complex regulatory networks with multiple pathways and feedbackcontrols. miRNAs are integral parts of these feedback and regulatorycircuits and can help regulate gene expression by keeping proteinproduction within limits (Herranz, H. & Cohen, S. M. Genes Dev 24,1339-1344 (2010); Posadas, D. M. & Carthew, R. W. Curr Opin Genet Dev27, 1-6 (2014)).

miRNA are also important in a large number of human diseases that areassociated with abnormal miRNA expression. This association underscoresthe importance of the miRNA regulatory pathway. Recent miRNA deletionstudies have linked miRNA with regulation of the immune responses(Stem-Ginossar, N. et al., Science 317, 376-381 (2007)).

miRNA also have a strong link to cancer and may play a role in differenttypes of cancer. miRNAs have been found to be downregulated in a numberof tumors. miRNA are important in the regulation of key cancer-relatedpathways, such as cell cycle control and the DNA damage response, andare therefore used in diagnosis and are being targeted clinically.MicroRNAs delicately regulate the balance of angiogenesis, such thatexperiments depleting all microRNAs suppresses tumor angiogenesis (Chen,S. et al., Genes Dev 28, 1054-1067 (2014)).

As has been shown for protein coding genes, miRNA genes are also subjectto epigenetic changes occurring with cancer. Many miRNA loci areassociated with CpG islands increasing their opportunity for regulationby DNA methylation (Weber, B., Stresemann, C., Brueckner, B. & Lyko, F.Cell Cycle 6, 1001-1005 (2007)). The majority of studies have usedtreatment with chromatin remodeling drugs to reveal epigeneticallysilenced miRNAs.

In addition to their role in RNA silencing, miRNA can also activatetranslation (Posadas, D. M. & Carthew, R. W. Curr Opin Genet Dev 27, 1-6(2014)). Knocking out these sites may lead to decreased expression ofthe targeted gene, while introducing these sites may increaseexpression.

Individual miRNA can be knocked out most effectively by mutating theseed sequence (bases 2-8 of the microRNA), which is important forbinding specificity. Cleavage in this region, followed by mis-repair byNHEJ can effectively abolish miRNA function by blocking binding totarget sites. miRNA could also be inhibited by specific targeting of thespecial loop region adjacent to the palindromic sequence. Catalyticallyinactive Cas9 can also be used to inhibit shRNA expression (Zhao, Y. etal., Sci Rep 4, 3943 (2014)). In addition to targeting the miRNA, thebinding sites can also be targeted and mutated to prevent the silencingby miRNA.

Human Cells

For ameliorating AATD, as described and illustrated herein, theprincipal targets for gene editing are human cells. For example, in theex vivo methods, the human cells are somatic cells, which after beingmodified using the techniques as described, can give rise to hepatocytesor progenitor cells. For example, in the in vivo methods, the humancells are hepatocytes, renal cells or cells from other affected organs.

By performing gene editing in autologous cells that are derived from andtherefore already completely matched with the patient in need, it ispossible to generate cells that can be safely re-introduced into thepatient, and effectively give rise to a population of cells that will beeffective in ameliorating one or more clinical conditions associatedwith the patient's disease.

Progenitor cells (also referred to as stem cells herein) are capable ofboth proliferation and giving rise to more progenitor cells, these inturn having the ability to generate a large number of mother cells thatcan in turn give rise to differentiated or differentiable daughtercells. The daughter cells themselves can be induced to proliferate andproduce progeny that subsequently differentiate into one or more maturecell types, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers then, to a cellwith the capacity or potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retains the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In one embodiment, the termprogenitor or stem cell refers to a generalized mother cell whosedescendants (progeny) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell may derive from amultipotent cell that itself is derived from a multipotent cell, and soon. While each of these multipotent cells may be considered stem cells,the range of cell types that each can give rise to may varyconsiderably. Some differentiated cells also have the capacity to giverise to cells of greater developmental potential. Such capacity may benatural or may be induced artificially upon treatment with variousfactors. In many biological instances, stem cells are also “multipotent”because they can produce progeny of more than one distinct cell type,but this is not required for “stem-ness.”

Self-renewal is another important aspect of the stem cell. In theory,self-renewal can occur by either of two major mechanisms. Stem cells maydivide asymmetrically, with one daughter retaining the stem state andthe other daughter expressing some distinct other specific function andphenotype. Alternatively, some of the stem cells in a population candivide symmetrically into two stems, thus maintaining some stem cells inthe population as a whole, while other cells in the population give riseto differentiated progeny only. Generally, “progenitor cells” have acellular phenotype that is more primitive (i.e., is at an earlier stepalong a developmental pathway or progression than is a fullydifferentiated cell). Often, progenitor cells also have significant orvery high proliferative potential. Progenitor cells can give rise tomultiple distinct differentiated cell types or to a singledifferentiated cell type, depending on the developmental pathway and onthe environment in which the cells develop and differentiate.

In the context of cell ontogeny, the adjective “differentiated,” or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellto which it is being compared. Thus, stem cells can differentiate intolineage-restricted precursor cells (such as a myocyte progenitor cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as a myocyte precursor), and then to anend-stage differentiated cell, such as a myocyte, which plays acharacteristic role in a certain tissue type, and may or may not retainthe capacity to proliferate further.

Induced Pluripotent Stem Cells

In some embodiments, the genetically engineered human cells describedherein are induced pluripotent stem cells (iPSCs). An advantage of usingiPSCs is that the cells can be derived from the same subject to whichthe progenitor cells are to be administered. That is, a somatic cell canbe obtained from a subject, reprogrammed to an induced pluripotent stemcell, and then re-differentiated into a progenitor cell to beadministered to the subject (e.g., autologous cells). Because theprogenitors are essentially derived from an autologous source, the riskof engraftment rejection or allergic response is reduced compared to theuse of cells from another subject or group of subjects. In addition, theuse of iPSCs negates the need for cells obtained from an embryonicsource. Thus, in one embodiment, the stem cells used in the disclosedmethods are not embryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to iPSCs. Exemplary methods are known to those of skill inthe art and are described briefly herein below.

The term “reprogramming” refers to a process that alters or reverses thedifferentiation state of a differentiated cell (e.g., a somatic cell).Stated another way, reprogramming refers to a process of driving thedifferentiation of a cell backwards to a more undifferentiated or moreprimitive type of cell. It should be noted that placing many primarycells in culture can lead to some loss of fully differentiatedcharacteristics. Thus, simply culturing such cells included in the termdifferentiated cells does not render these cells non-differentiatedcells (e.g., undifferentiated cells) or pluripotent cells. Thetransition of a differentiated cell to pluripotency requires areprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming. In some embodiments,reprogramming encompasses complete reversion of the differentiationstate of a differentiated cell (e.g., a somatic cell) to a pluripotentstate or a multipotent state. In some embodiments, reprogrammingencompasses complete or partial reversion of the differentiation stateof a differentiated cell (e.g., a somatic cell) to an undifferentiatedcell (e.g., an embryonic-like cell). Reprogramming can result inexpression of particular genes by the cells, the expression of whichfurther contributes to reprogramming. In certain embodiments describedherein, reprogramming of a differentiated cell (e.g., a somatic cell)causes the differentiated cell to assume an undifferentiated state(e.g., is an undifferentiated cell). The resulting cells are referred toas “reprogrammed cells,” or “induced pluripotent stem cells (iPSCs oriPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a myogenic stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some embodiments.

Many methods are known in the art that can be used to generatepluripotent stem cells from somatic cells. Any such method thatreprograms a somatic cell to the pluripotent phenotype would beappropriate for use in the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been described. Mousesomatic cells can be converted to ES cell-like cells with expandeddevelopmental potential by the direct transduction of Oct4, Sox2, Klf4,and c-Myc; see, e.g., Takahashi and Yamanaka, Cell 126(4): 663-76(2006). iPSCs resemble ES cells, as they restore thepluripotency-associated transcriptional circuitry and much of theepigenetic landscape. In addition, mouse iPSCs satisfy all the standardassays for pluripotency: specifically, in vitro differentiation intocell types of the three germ layers, teratoma formation, contribution tochimeras, germline transmission [see, e.g., Maherali and Hochedlinger,Cell Stem Cell. 3(6):595-605 (2008)], and tetraploid complementation.

Human iPSCs can be obtained using similar transduction methods, and thetranscription factor trio, OCT4, SOX2, and NANOG, has been establishedas the core set of transcription factors that govern pluripotency; see,e.g., Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57(2014); Barrett et al., Stem Cells Trans Med 3:1-6 sctm.2014-0121(2014); Focosi et al., Blood Cancer Journal 4: e211 (2014); andreferences cited therein. The production of iPSCs can be achieved by theintroduction of nucleic acid sequences encoding stem cell-associatedgenes into an adult, somatic cell, historically using viral vectors.

iPSCs can be generated or derived from terminally differentiated somaticcells, as well as from adult stem cells, or somatic stem cells. That is,a non-pluripotent progenitor cell can be rendered pluripotent ormultipotent by reprogramming. In such instances, it may not be necessaryto include as many reprogramming factors as required to reprogram aterminally differentiated cell. Further, reprogramming can be induced bythe non-viral introduction of reprogramming factors, e.g., byintroducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 7(5):618-30 (2010). Reprogramming can beachieved by introducing a combination of nucleic acids encoding stemcell-associated genes, including, for example, Oct-4 (also known asOct-¾ or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28. In oneembodiment, reprogramming using the methods and compositions describedherein can further comprise introducing one or more of Oct-¾, a memberof the Sox family, a member of the Klf family, and a member of the Mycfamily to a somatic cell. In one embodiment, the methods andcompositions described herein further comprise introducing one or moreof each of Oct-4, Sox2, Nanog, c-MYC and Klf4 for reprogramming. Asnoted above, the exact method used for reprogramming is not necessarilycritical to the methods and compositions described herein. However,where cells differentiated from the reprogrammed cells are to be usedin, e.g., human therapy, in one embodiment the reprogramming is noteffected by a method that alters the genome. Thus, in such embodiments,reprogramming is achieved, e.g., without the use of viral or plasmidvectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various agents, e.g., small molecules, as shown by Shi etal., Cell-Stem Cell 2:525-528 (2008); Huangfu et al., NatureBiotechnology 26(7):795-797 (2008) and Marson et al., Cell-Stem Cell 3:132-135 (2008). Thus, an agent or combination of agents that enhance theefficiency or rate of induced pluripotent stem cell production can beused in the production of patient-specific or disease-specific iPSCs.Some non-limiting examples of agents that enhance reprogrammingefficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9ahistone methyltransferase), PD0325901 (a MEK inhibitor), DNAmethyltransferase inhibitors, histone deacetylase (HDAC) inhibitors,valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide,hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-CI-UCHA (e.g.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Other reprogrammingenhancing agents include, for example, dominant negative forms of theHDACs (e.g., catalytically inactive forms), siRNA inhibitors of theHDACs, and antibodies that specifically bind to the HDACs. Suchinhibitors are available, e.g., from BIOMOL International, Fukasawa,Merck Biosciences, Novartis, Gloucester Pharmaceuticals, TitanPharmaceuticals, MethylGene, and Sigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers are selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto,Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. In one embodiment, a cellthat expresses Oct4 or Nanog is identified as pluripotent. Methods fordetecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses. In someembodiments, detection involves not only RT-PCR, but also includesdetection of protein markers. Intracellular markers may be bestidentified via RT-PCR, or protein detection methods such asimmunocytochemistry, while cell surface markers are readily identified,e.g., by immunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate into cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells are introduced into nude mice and histologyand/or immunohistochemistry is performed on a tumor arising from thecells. The growth of a tumor comprising cells from all three germlayers, for example, further indicates that the cells are pluripotentstem cells.

Hepatocytes

In some embodiments, the genetically engineered human cells describedherein are hepatocytes. A hepatocyte is a cell of the main parenchymaltissue of the liver. Hepatocytes make up 70-85% of the liver's mass.These cells are involved in: protein synthesis; protein storage;transformation of carbohydrates; synthesis of cholesterol, bile saltsand phospholipids; detoxification, modification, and excretion ofexogenous and endogenous substances; and initiation of formation andsecretion of bile.

SERPINA1 is primarily expressed in hepatocytes (parenchymal livercells), which are a major source of circulating protein, with secondaryexpression in monocytes and neutrophils. Therefore, the correction ofSERPINA1 would be primarily targeted at hepatocytes and the liver.

Creating Patient Specific IPSCs

One step of the ex vivo methods of the invention involves creating apatient specific iPS cell, patient specific iPS cells, or a patientspecific iPS cell line. There are many established methods in the artfor creating patient specific iPS cells, as described in Takahashi andYamanaka 2006; Takahashi, Tanabe et al. 2007. For example, the creatingstep comprises: a) isolating a somatic cell, such as a skin cell orfibroblast from the patient; and b) introducing a set ofpluripotency-associated genes into the somatic cell in order to inducethe cell to become a pluripotent stem cell. In some embodiments, the setof pluripotency-associated genes is one or more of the genes selectedfrom the group consisting of OCT4, SOX2, KLF4, Lin28, NANOG, and cMYC.

Performing a Biopsy or Aspirate of the Patient's Liver or Bone Marrow

A biopsy or aspirate is a sample of tissue or fluid taken from the body.There are many different kinds of biopsies or aspirates. Nearly all ofthem involve using a sharp tool to remove a small amount of tissue. Ifthe biopsy will be on the skin or other sensitive area, numbing medicineis applied first. A biopsy or aspirate may be performed according to anyof the known methods in the art. For example, in a liver biopsy, aneedle is injected into the liver through the skin of the belly,capturing the liver tissue. For example, in a bone marrow aspirate, alarge needle is used to enter the pelvis bone to collect bone marrow.

Isolating a Liver Specific Progenitor Cell or Primary Hepatocyte

Liver specific progenitor cells and primary hepatocytes may be isolatedaccording to any method known in the art. For example, human hepatocytesare isolated from fresh surgical specimens (e.g., an autologous sample).Healthy liver tissue is used to isolate hepatocytes by collagenasedigestion. The obtained cell suspension is filtered through a 100-mmnylon mesh and sedimented by centrifugation at 50 g for 5 minutes,resuspended, and washed two to three times in cold wash medium. Humanliver stem cells are obtained by culturing under stringent conditions ofhepatocytes obtained from fresh liver preparations. Hepatocytes seededon collagen-coated plates are cultured for 2 weeks. After 2 weeks,surviving cells are removed, and characterized for expression of stemcells markers (Herrera et al, STEM CELLS 2006; 24: 2840-2850).

Isolating a Mesenchymal Stem Cell

Mesenchymal stem cells may be isolated according to any method known inthe art, such as from a patient's bone marrow or peripheral blood. Forexample, marrow aspirate is collected into a syringe with heparin. Cellsare washed and centrifuged on a Percoll™. Cells, such as blood cells,liver cells, interstitial cells, macrophages, mast cells, andthymocytes, are separated using Percoll™. The cells are cultured inDulbecco's modified Eagle's medium (DMEM) (low glucose) containing 10%fetal bovine serum (FBS) (Pittinger M F, Mackay A M, Beck S C et al.,Science 1999; 284:143-147).

Genome Editing

Genome editing generally refers to the process of modifying thenucleotide sequence of a genome, preferably in a precise orpre-determined manner. Examples of methods of genome editing describedherein include methods of using site-directed nucleases to cutdeoxyribonucleic acid (DNA) at precise target locations in the genome,thereby creating double-strand or single-strand DNA breaks at particularlocations within the genome. Such breaks can be and regularly arerepaired by natural, endogenous cellular processes, such ashomology-directed repair (HDR) and non-homologous end-joining (NHEJ), asrecently reviewed in Cox et al., Nature Medicine 21(2), 121-31 (2015).These two main DNA repair processes consist of a family of alternativepathways. NHEJ directly joins the DNA ends resulting from adouble-strand break, sometimes with the loss or addition of nucleotidesequence, which may disrupt or enhance gene expression. HDR utilizes ahomologous sequence, or donor sequence, as a template for inserting adefined DNA sequence at the break point. The homologous sequence may bein the endogenous genome, such as a sister chromatid. Alternatively, thedonor may be an exogenous nucleic acid, such as a plasmid, asingle-strand oligonucleotide, a double-stranded oligonucleotide, aduplex oligonucleotide or a virus, that has regions of high homologywith the nuclease-cleaved locus, but which may also contain additionalsequence or sequence changes including deletions that can beincorporated into the cleaved target locus. A third repair mechanism ismicrohomology-mediated end joining (MMEJ), also referred to as“Alternative NHEJ”, in which the genetic outcome is similar to NHEJ inthat small deletions and insertions can occur at the cleavage site. MMEJmakes use of homologous sequences of a few basepairs flanking the DNAbreak site to drive a more favored DNA end joining repair outcome, andrecent reports have further elucidated the molecular mechanism of thisprocess; see, e.g., Cho and Greenberg, Nature 518, 174-76 (2015); Kentet al., Nature Structural and Molecular Biology, Adv. Onlinedoi:10.1038/nsmb.2961 (2015); Mateos-Gomez et al., Nature 518, 254-57(2015); Ceccaldi et al., Nature 528, 258-62 (2015). In some instances itmay be possible to predict likely repair outcomes based on analysis ofpotential microhomologies at the site of the DNA break.

Each of these genome editing mechanisms can be used to create desiredgenomic alterations. A step in the genome editing process is to createone or two DNA breaks, the latter as double-strand breaks or as twosingle-stranded breaks, in the target locus as close as possible to thesite of intended mutation. This can be achieved via the use ofsite-directed polypeptides, as described and illustrated herein.

Site-directed polypeptides, such as a DNA endonuclease, can introducedouble-strand breaks or single-strand breaks in nucleic acids, e.g.,genomic DNA. The double-strand break can stimulate a cell's endogenousDNA-repair pathways (e.g., homology-dependent repair or non-homologousend joining or alternative non-homologous end joining (A-NHEJ) ormicrohomology-mediated end joining). NHEJ can repair cleaved targetnucleic acid without the need for a homologous template. This cansometimes result in small deletions or insertions (indels) in the targetnucleic acid at the site of cleavage, and can lead to disruption oralteration of gene expression. HDR can occur when a homologous repairtemplate, or donor, is available. The homologous donor templatecomprises sequences that are homologous to sequences flanking the targetnucleic acid cleavage site. The sister chromatid is generally used bythe cell as the repair template. However, for the purposes of genomeediting, the repair template is often supplied as an exogenous nucleicacid, such as a plasmid, duplex oligonucleotide, single-strandoligonucleotide, double-stranded oligonucleotide, or viral nucleic acid.With exogenous donor templates, it is common to introduce an additionalnucleic acid sequence (such as a transgene) or modification (such as asingle or multiple base change or a deletion) between the flankingregions of homology so that the additional or altered nucleic acidsequence also becomes incorporated into the target locus. MMEJ resultsin a genetic outcome that is similar to NHEJ in that small deletions andinsertions can occur at the cleavage site. MMEJ makes use of homologoussequences of a few basepairs flanking the cleavage site to drive afavored end-joining DNA repair outcome. In some instances it may bepossible to predict likely repair outcomes based on analysis ofpotential microhomologies in the nuclease target regions.

Thus, in some cases, homologous recombination is used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a donorpolynucleotide (or donor or donor sequence or polynucleotide donortemplate) herein. In some embodiments, the donor polynucleotide, aportion of the donor polynucleotide, a copy of the donor polynucleotide,or a portion of a copy of the donor polynucleotide is inserted into thetarget nucleic acid cleavage site. In some embodiments, the donorpolynucleotide is an exogenous polynucleotide sequence, i.e., a sequencethat does not naturally occur at the target nucleic acid cleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to,for example, mutations, deletions, alterations, integrations, genecorrection, gene replacement, gene tagging, transgene insertion,nucleotide deletion, gene disruption, translocations and/or genemutation. The processes of deleting genomic DNA and integratingnon-native nucleic acid into genomic DNA are examples of genome editing.

CRISPR Endonuclease System

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)genomic locus can be found in the genomes of many prokaryotes (e.g.,bacteria and archaea). In prokaryotes, the CRISPR locus encodes productsthat function as a type of immune system to help defend the prokaryotesagainst foreign invaders, such as virus and phage. There are threestages of CRISPR locus function: integration of new sequences into theCRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreigninvader nucleic acid. Five types of CRISPR systems (e.g., Type I, TypeII, Type III, Type U, and Type V) have been identified.

A CRISPR locus includes a number of short repeating sequences referredto as “repeats.” When expressed, the repeats can form secondary hairpinstructures (e.g., hairpins) and/or comprise unstructured single-strandedsequences. The repeats usually occur in clusters and frequently divergebetween species. The repeats are regularly interspaced with uniqueintervening sequences referred to as “spacers,” resulting in arepeat-spacer-repeat locus architecture. The spacers are identical to orhave high homology with known foreign invader sequences. A spacer-repeatunit encodes a crisprRNA (crRNA), which is processed into a mature formof the spacer-repeat unit. A crRNA comprises a “seed” or spacer sequencethat is involved in targeting a target nucleic acid (in the naturallyoccurring form in prokaryotes, the spacer sequence targets the foreigninvader nucleic acid). A spacer sequence is located at the 5′ or 3′ endof the crRNA.

A CRISPR locus also comprises polynucleotide sequences encoding CRISPRAssociated (Cas) genes. Cas genes encode endonucleases involved in thebiogenesis and the interference stages of crRNA function in prokaryotes.Some Cas genes comprise homologous secondary and/or tertiary structures.

Type II CRISPR Systems

crRNA biogenesis in a Type II CRISPR system in nature requires atrans-activating CRISPR RNA (tracrRNA). The tracrRNA is modified byendogenous RNaseIII, and then hybridizes to a crRNA repeat in thepre-crRNA array. Endogenous RNaseIII is recruited to cleave thepre-crRNA. Cleaved crRNAs are subjected to exoribonuclease trimming toproduce the mature crRNA form (e.g., 5′ trimming). The tracrRNA remainshybridized to the crRNA, and the tracrRNA and the crRNA associate with asite-directed polypeptide (e.g., Cas9). The crRNA of thecrRNA-tracrRNA-Cas9 complex guides the complex to a target nucleic acidto which the crRNA can hybridize. Hybridization of the crRNA to thetarget nucleic acid activates Cas9 for targeted nucleic acid cleavage.The target nucleic acid in a Type II CRISPR system is referred to as aprotospacer adjacent motif (PAM). In nature, the PAM is essential tofacilitate binding of a site-directed polypeptide (e.g., Cas9) to thetarget nucleic acid. Type II systems (also referred to as Nmeni orCASS4) are further subdivided into Type II-A (CASS4) and II-B (CASS4a).Jinek et al., Science, 337(6096):816-821 (2012) showed that theCRISPR/Cas9 system is useful for RNA-programmable genome editing, andinternational patent application publication number WO2013/176772provides numerous examples and applications of the CRISPR/Casendonuclease system for site-specific gene editing.

Type V CRISPR Systems

Type V CRISPR systems have several important differences from Type IIsystems. For example, Cpf1 is a single RNA-guided endonuclease that, incontrast to Type II systems, lacks tracrRNA. In fact, Cpf1-associatedCRISPR arrays are processed into mature crRNAS without the requirementof an additional trans-activating tracrRNA. The Type V CRISPR array isprocessed into short mature crRNAs of 42-44 nucleotides in length, witheach mature crRNA beginning with 19 nucleotides of direct repeatfollowed by 23-25 nucleotides of spacer sequence. In contrast, maturecrRNAs in Type II systems start with 20-24 nucleotides of spacersequence followed by about 22 nucleotides of direct repeat. Also, Cpf1utilizes a T-rich protospacer-adjacent motif such that Cpf1-crRNAcomplexes efficiently cleave target DNA preceded by a short T-rich PAM,which is in contrast to the G-rich PAM following the target DNA for TypeII systems. Thus, Type V systems cleave at a point that is distant fromthe PAM, while Type II systems cleave at a point that is adjacent to thePAM. In addition, in contrast to Type II systems, Cpf1 cleaves DNA via astaggered DNA double-stranded break with a 4 or 5 nucleotide 5′overhang. Type II systems cleave via a blunt double-stranded break.Similar to Type II systems, Cpf1 contains a predicted RuvC-likeendonuclease domain, but lacks a second HNH endonuclease domain, whichis in contrast to Type II systems.

Cas Genes/Polypeptides and Protospacer Adjacent Motifs

Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides in FIG.1 of Fonfara et al., Nucleic Acids Research, 42: 2577-2590 (2014). TheCRISPR/Cas gene naming system has undergone extensive rewriting sincethe Cas genes were discovered. FIG. 5 of Fonfara, supra, provides PAMsequences for the Cas9 polypeptides from various species.

Site-Directed Polypeptides

A site-directed polypeptide is a nuclease used in genome editing tocleave DNA. The site-directed may be administered to a cell or a patientas either one or more polypeptides, or one or more mRNAs encoding thepolypeptide.

In the context of a CRISPR/Cas or CRISPR/Cpf1 system, the site-directedpolypeptide can bind to a guide RNA that, in turn, specifies the site inthe target DNA to which the polypeptide is directed. In embodiments ofCRISPR/Cas or CRISPR/Cpf1 systems herein, the site-directed polypeptideis an endonuclease, such as a DNA endonuclease.

In some embodiments, a site-directed polypeptide comprises a pluralityof nucleic acid-cleaving (i.e., nuclease) domains. Two or more nucleicacid-cleaving domains can be linked together via a linker. In someembodiments, the linker comprises a flexible linker. Linkers maycomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or more amino acids in length.

Naturally-occurring wild-type Cas9 enzymes comprise two nucleasedomains, a HNH nuclease domain and a RuvC domain. Herein, the “Cas9”refers to both naturally-occurring and recombinant Cas9s. Cas9 enzymescontemplated herein comprises a HNH or HNH-like nuclease domain, and/ora RuvC or RuvC-like nuclease domain.

HNH or HNH-like domains comprise a McrA-like fold. HNH or HNH-likedomains comprises two antiparallel β-strands and an α-helix. HNH orHNH-like domains comprises a metal binding site (e.g., a divalent cationbinding site). HNH or HNH-like domains can cleave one strand of a targetnucleic acid (e.g., the complementary strand of the crRNA targetedstrand).

RuvC or RuvC-like domains comprise an RNaseH or RNaseH-like fold.RuvC/RNaseH domains are involved in a diverse set of nucleic acid-basedfunctions including acting on both RNA and DNA. The RNaseH domaincomprises 5 β-strands surrounded by a plurality of α-helices.RuvC/RNaseH or RuvC/RNaseH-like domains comprise a metal binding site(e.g., a divalent cation binding site). RuvC/RNaseH or RuvC/RNaseH-likedomains can cleave one strand of a target nucleic acid (e.g., thenon-complementary strand of a double-stranded target DNA).

Site-directed polypeptides can introduce double-strand breaks orsingle-strand breaks in nucleic acids, e.g., genomic DNA. Thedouble-strand break can stimulate a cell's endogenous DNA-repairpathways (e.g., homology-dependent repair (HDR) or non-homologous endjoining (NHEJ) or alternative non-homologous end joining (A-NHEJ) ormicrohomology-mediated end joining (MMEJ)). NHEJ can repair cleavedtarget nucleic acid without the need for a homologous template. This cansometimes result in small deletions or insertions (indels) in the targetnucleic acid at the site of cleavage, and can lead to disruption oralteration of gene expression. HDR can occur when a homologous repairtemplate, or donor, is available. The homologous donor templatecomprises sequences that are homologous to sequences flanking the targetnucleic acid cleavage site. The sister chromatid is generally used bythe cell as the repair template. However, for the purposes of genomeediting, the repair template is often supplied as an exogenous nucleicacid, such as a plasmid, duplex oligonucleotide, single-strandoligonucleotide or viral nucleic acid. With exogenous donor templates,it is common to introduce an additional nucleic acid sequence (such as atransgene) or modification (such as a single or multiple base change ora deletion) between the flanking regions of homology so that theadditional or altered nucleic acid sequence also becomes incorporatedinto the target locus. MMEJ results in a genetic outcome that is similarto NHEJ in that small deletions and insertions can occur at the cleavagesite. MMEJ makes use of homologous sequences of a few basepairs flankingthe cleavage site to drive a favored end-joining DNA repair outcome. Insome instances it may be possible to predict likely repair outcomesbased on analysis of potential microhomologies in the nuclease targetregions.

Thus, in some cases, homologous recombination is used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a donorpolynucleotide (or donor or donor sequence) herein. In some embodiments,the donor polynucleotide, a portion of the donor polynucleotide, a copyof the donor polynucleotide, or a portion of a copy of the donorpolynucleotide is inserted into the target nucleic acid cleavage site.In some embodiments, the donor polynucleotide is an exogenouspolynucleotide sequence, i.e., a sequence that does not naturally occurat the target nucleic acid cleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to,for example, mutations, deletions, alterations, integrations, genecorrection, gene replacement, gene tagging, transgene insertion,nucleotide deletion, gene disruption, translocations and/or genemutation. The processes of deleting genomic DNA and integratingnon-native nucleic acid into genomic DNA are examples of genome editing.

In some embodiments, the site-directed polypeptide comprises an aminoacid sequence having at least 10%, at least 15%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% amino acid sequence identity to a wild-type exemplarysite-directed polypeptide [e.g., Cas9 from S. pyogenes, US2014/0068797Sequence ID No. 8 or Sapranauskas et al., Nucleic Acids Res, 39(21):9275-9282 (2011)], and various other site-directed polypeptides).

In some embodiments, the site-directed polypeptide comprises an aminoacid sequence having at least 10%, at least 15%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least99%, or 100% amino acid sequence identity to the nuclease domain of awild-type exemplary site-directed polypeptide (e.g., Cas9 from S.pyogenes, supra).

In some embodiments, a site-directed polypeptide comprises at least 70,75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids. In some embodiments, a site-directed polypeptidecomprises at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids. In some embodiments, a site-directedpolypeptide comprises at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100%identity to a wild-type site-directed polypeptide (e.g., Cas9 from S.pyogenes, supra) over 10 contiguous amino acids in a HNH nuclease domainof the site-directed polypeptide. In some embodiments, a site-directedpolypeptide comprises at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100%identity to a wild-type site-directed polypeptide (e.g., Cas9 from S.pyogenes, supra) over 10 contiguous amino acids in a HNH nuclease domainof the site-directed polypeptide. In some embodiments, a site-directedpolypeptide comprises at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100%identity to a wild-type site-directed polypeptide (e.g., Cas9 from S.pyogenes, supra) over 10 contiguous amino acids in a RuvC nucleasedomain of the site-directed polypeptide. In some embodiments, asite-directed polypeptide comprises at most: 70, 75, 80, 85, 90, 95, 97,99, or 100% identity to a wild-type site-directed polypeptide (e.g.,Cas9 from S. pyogenes, supra) over 10 contiguous amino acids in a RuvCnuclease domain of the site-directed polypeptide.

In some embodiments, the site-directed polypeptide comprises a modifiedform of a wild-type exemplary site-directed polypeptide. The modifiedform of the wild-type exemplary site-directed polypeptide comprises amutation that reduces the nucleic acid-cleaving activity of thesite-directed polypeptide. In some embodiments, the modified form of thewild-type exemplary site-directed polypeptide has less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, or less than1% of the nucleic acid-cleaving activity of the wild-type exemplarysite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra). Themodified form of the site-directed polypeptide can have no substantialnucleic acid-cleaving activity. When a site-directed polypeptide is amodified form that has no substantial nucleic acid-cleaving activity, itis referred to herein as “enzymatically inactive.”

In some embodiments, the modified form of the site-directed polypeptidecomprises a mutation such that it can induce a single-strand break (SSB)on a target nucleic acid (e.g., by cutting only one of thesugar-phosphate backbones of a double-strand target nucleic acid). Insome embodiments, the mutation results in less than 90%, less than 80%,less than 70%, less than 60%, less than 50%, less than 40%, less than30%, less than 20%, less than 10%, less than 5%, or less than 1% of thenucleic acid-cleaving activity in one or more of the plurality ofnucleic acid-cleaving domains of the wild-type site directed polypeptide(e.g., Cas9 from S. pyogenes, supra). In some embodiments, the mutationresults in one or more of the plurality of nucleic acid-cleaving domainsretaining the ability to cleave the complementary strand of the targetnucleic acid, but reducing its ability to cleave the non-complementarystrand of the target nucleic acid. In some embodiments, the mutationresults in one or more of the plurality of nucleic acid-cleaving domainsretaining the ability to cleave the non-complementary strand of thetarget nucleic acid, but reducing its ability to cleave thecomplementary strand of the target nucleic acid. For example, residuesin the wild-type exemplary S. pyogenes Cas9 polypeptide, such as Asp10,His840, Asn854 and Asn856, are mutated to inactivate one or more of theplurality of nucleic acid-cleaving domains (e.g., nuclease domains). Insome embodiments, the residues to be mutated correspond to residuesAsp10, His840, Asn854 and Asn856 in the wild-type exemplary S. pyogenesCas9 polypeptide (e.g., as determined by sequence and/or structuralalignment). Non-limiting examples of mutations include D10A, H840A,N854A or N856A. One skilled in the art will recognize that mutationsother than alanine substitutions are suitable.

In some embodiments, a D10A mutation is combined with one or more ofH840A, N854A, or N856A mutations to produce a site-directed polypeptidesubstantially lacking DNA cleavage activity. In some embodiments, aH840A mutation is combined with one or more of D10A, N854A, or N856Amutations to produce a site-directed polypeptide substantially lackingDNA cleavage activity. In some embodiments, a N854A mutation is combinedwith one or more of H840A, D10A, or N856A mutations to produce asite-directed polypeptide substantially lacking DNA cleavage activity.In some embodiments, a N856A mutation is combined with one or more ofH840A, N854A, or D10A mutations to produce a site-directed polypeptidesubstantially lacking DNA cleavage activity. Site-directed polypeptidesthat comprise one substantially inactive nuclease domain are referred toas “nickases”.

Nickase variants of RNA-guided endonucleases, for example Cas9, can beused to increase the specificity of CRISPR-mediated genome editing. Wildtype Cas9 is typically guided by a single guide RNA designed tohybridize with a specified ˜20 nucleotide sequence in the targetsequence (such as an endogenous genomic locus). However, severalmismatches can be tolerated between the guide RNA and the target locus,effectively reducing the length of required homology in the target siteto, for example, as little as 13 nt of homology, and thereby resultingin elevated potential for binding and double-strand nucleic acidcleavage by the CRISPR/Cas9 complex elsewhere in the target genome—alsoknown as off-target cleavage. Because nickase variants of Cas9 each onlycut one strand, in order to create a double-strand break it is necessaryfor a pair of nickases to bind in close proximity and on oppositestrands of the target nucleic acid, thereby creating a pair of nicks,which is the equivalent of a double-strand break. This requires that twoseparate guide RNAs—one for each nickase—must bind in close proximityand on opposite strands of the target nucleic acid. This requirementessentially doubles the minimum length of homology needed for thedouble-strand break to occur, thereby reducing the likelihood that adouble-strand cleavage event will occur elsewhere in the genome, wherethe two guide RNA sites—if they exist—are unlikely to be sufficientlyclose to each other to enable the double-strand break to form. Asdescribed in the art, nickases can also be used to promote HDR versusNHEJ. HDR can be used to introduce selected changes into target sites inthe genome through the use of specific donor sequences that effectivelymediate the desired changes. Descriptions of various CRISPR/Cas systemsfor use in gene editing can be found, e.g., in international patentapplication publication number WO2013/176772, and in NatureBiotechnology 32, 347-355 (2014), and references cited therein.

Mutations contemplated include substitutions, additions, and deletions,or any combination thereof. In some embodiments, the mutation convertsthe mutated amino acid to alanine. In some embodiments, the mutationconverts the mutated amino acid to another amino acid (e.g., glycine,serine, threonine, cysteine, valine, leucine, isoleucine, methionine,proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamicacid, asparagines, glutamine, histidine, lysine, or arginine). In someembodiments, the mutation converts the mutated amino acid to anon-natural amino acid (e.g., selenomethionine). In some embodiments,the mutation converts the mutated amino acid to amino acid mimics (e.g.,phosphomimics). In some embodiments, the mutation is a conservativemutation. For example, the mutation can convert the mutated amino acidto amino acids that resemble the size, shape, charge, polarity,conformation, and/or rotamers of the mutated amino acids (e.g.,cysteine/serine mutation, lysine/asparagine mutation,histidine/phenylalanine mutation). In some embodiments, the mutationcauses a shift in reading frame and/or the creation of a premature stopcodon. In some embodiments, mutations cause changes to regulatoryregions of genes or loci that affect expression of one or more genes.

In some embodiments, the site-directed polypeptide (e.g., variant,mutated, enzymatically inactive and/or conditionally enzymaticallyinactive site-directed polypeptide) targets nucleic acid. In someembodiments, the site-directed polypeptide (e.g., variant, mutated,enzymatically inactive and/or conditionally enzymatically inactiveendoribonuclease) targets DNA. In some embodiments, the site-directedpolypeptide (e.g., variant, mutated, enzymatically inactive and/orconditionally enzymatically inactive endoribonuclease) targets RNA.

In some embodiments, the site-directed polypeptide comprises one or morenon-native sequences (e.g., the site-directed polypeptide is a fusionprotein).

In some embodiments, the site-directed polypeptide comprises an aminoacid sequence comprising at least 15% amino acid identity to a Cas9 froma bacterium (e.g., S. pyogenes), a nucleic acid binding domain, and twonucleic acid cleaving domains (i.e., a HNH domain and a RuvC domain).

In some embodiments, the site-directed polypeptide comprises an aminoacid sequence comprising at least 15% amino acid identity to a Cas9 froma bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains(i.e., a HNH domain and a RuvC domain).

In some embodiments, the site-directed polypeptide comprises an aminoacid sequence comprising at least 15% amino acid identity to a Cas9 froma bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains,wherein one or both of the nucleic acid cleaving domains comprise atleast 50% amino acid identity to a nuclease domain from Cas9 from abacterium (e.g., S. pyogenes).

In some embodiments, the site-directed polypeptide comprises an aminoacid sequence comprising at least 15% amino acid identity to a Cas9 froma bacterium (e.g., S. pyogenes), two nucleic acid cleaving domains(i.e., a HNH domain and a RuvC domain), and non-native sequence (forexample, a nuclear localization signal) or a linker linking thesite-directed polypeptide to a non-native sequence.

In some embodiments, the site-directed polypeptide comprises an aminoacid sequence comprising at least 15% amino acid identity to a Cas9 froma bacterium (e.g., S. pyogenes), two nucleic acid cleaving domains(i.e., a HNH domain and a RuvC domain), wherein the site-directedpolypeptide comprises a mutation in one or both of the nucleic acidcleaving domains that reduces the cleaving activity of the nucleasedomains by at least 50%.

In some embodiments, the site-directed polypeptide comprises an aminoacid sequence comprising at least 15% amino acid identity to a Cas9 froma bacterium (e.g., S. pyogenes), and two nucleic acid cleaving domains(i.e., a HNH domain and a RuvC domain), wherein one of the nucleasedomains comprises mutation of aspartic acid 10, and/or wherein one ofthe nuclease domains comprises mutation of histidine 840, and whereinthe mutation reduces the cleaving activity of the nuclease domain(s) byat least 50%.

In some embodiments of the invention, the one or more site-directedpolypeptides, e.g. DNA endonucleases, include two nickases that togethereffect one double-strand break at a specific locus in the genome, orfour nickases that together effect two double-strand breaks at specificloci in the genome. Alternatively, one site-directed polypeptide, e.g.DNA endonuclease, effects one double-strand break at a specific locus inthe genome.

Genome-Targeting Nucleic Acid

The present disclosure provides a genome-targeting nucleic acid that candirect the activities of an associated polypeptide (e.g., asite-directed polypeptide) to a specific target sequence within a targetnucleic acid. In some embodiments, the genome-targeting nucleic acid isan RNA. A genome-targeting RNA is referred to as a “guide RNA” or “gRNA”herein. A guide RNA comprises at least a spacer sequence that hybridizesto a target nucleic acid sequence of interest, and a CRISPR repeatsequence. In Type II systems, the gRNA also comprises a second RNAcalled the tracrRNA sequence. In the Type II guide RNA (gRNA), theCRISPR repeat sequence and tracrRNA sequence hybridize to each other toform a duplex. In the Type V guide RNA (gRNA), the crRNA forms a duplex.In both systems, the duplex binds a site-directed polypeptide, such thatthe guide RNA and site-direct polypeptide form a complex. Thegenome-targeting nucleic acid provides target specificity to the complexby virtue of its association with the site-directed polypeptide. Thegenome-targeting nucleic acid thus directs the activity of thesite-directed polypeptide.

Exemplary guide RNAs include the spacer sequences in SEQ ID NOs:1-68,297, for example, in SEQ ID NOs: 54,860-68,297, shown with thegenome location of their target sequence and the associated Cas9 or Cpf1cut site, wherein the genome location is based on the GRCh38/hg38 humangenome assembly. As is understood by the person of ordinary skill in theart, each guide RNA is designed to include a spacer sequencecomplementary to its genomic target sequence. For example, each of thespacer sequences in SEQ ID NOs: 1-68,297, for example, in SEQ ID NOs:54,860-68,297 may be put into a single RNA chimera or a crRNA (alongwith a corresponding tracrRNA). See Jinek et al., Science, 337, 816-821(2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

In some embodiments, the genome-targeting nucleic acid is adouble-molecule guide RNA. In some embodiments, the genome-targetingnucleic acid is a single-molecule guide RNA.

A double-molecule guide RNA comprises two strands of RNA. The firststrand comprises in the 5′ to 3′ direction, an optional spacer extensionsequence, a spacer sequence and a minimum CRISPR repeat sequence. Thesecond strand comprises a minimum tracrRNA sequence (complementary tothe minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence.

A single-molecule guide RNA (sgRNA) in a Type II system comprises, inthe 5′ to 3′ direction, an optional spacer extension sequence, a spacersequence, a minimum CRISPR repeat sequence, a single-molecule guidelinker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence. The optional tracrRNA extensionmay comprise elements that contribute additional functionality (e.g.,stability) to the guide RNA. The single-molecule guide linker links theminimum CRISPR repeat and the minimum tracrRNA sequence to form ahairpin structure. The optional tracrRNA extension comprises one or morehairpins.

A single-molecule guide RNA (sgRNA) in a Type V system comprises, in the5′ to 3′ direction, a minimum CRISPR repeat sequence and a spacersequence.

By way of illustration, guide RNAs used in the CRISPR/Cas system, orother smaller RNAs can be readily synthesized by chemical means, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach used for generating RNAs of greater length isto produce two or more molecules that are ligated together. Much longerRNAs, such as those encoding a Cas9 or Cpf1 endonuclease, are morereadily generated enzymatically. Various types of RNA modifications canbe introduced during or after chemical synthesis and/or enzymaticgeneration of RNAs, e.g., modifications that enhance stability, reducethe likelihood or degree of innate immune response, and/or enhance otherattributes, as described in the art.

Spacer Extension Sequence

In some embodiments of genome-targeting nucleic acids, a spacerextension sequence can provide stability and/or provide a location formodifications of a genome-targeting nucleic acid. A spacer extensionsequence can modify on- or off-target activity or specificity. In someembodiments, a spacer extension sequence is provided. A spacer extensionsequence may have a length of more than 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260,280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000,or 7000 or more nucleotides. A spacer extension sequence may have alength of less than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340,360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000, 7000 or morenucleotides. In some embodiments, a spacer extension sequence is lessthan 10 nucleotides in length. In some embodiments, a spacer extensionsequence is between 10-30 nucleotides in length. In some embodiments, aspacer extension sequence is between 30-70 nucleotides in length.

In some embodiments, the spacer extension sequence comprises anothermoiety (e.g., a stability control sequence, an endoribonuclease bindingsequence, a ribozyme). In some embodiments, the moiety decreases orincreases the stability of a nucleic acid targeting nucleic acid. Insome embodiments, the moiety is a transcriptional terminator segment(i.e., a transcription termination sequence). In some embodiments, themoiety functions in a eukaryotic cell. In some embodiments, the moietyfunctions in a prokaryotic cell. In some embodiments, the moietyfunctions in both eukaryotic and prokaryotic cells. Non-limitingexamples of suitable moieties include: a 5′ cap (e.g., a7-methylguanylate cap (m7 G)), a riboswitch sequence (e.g., to allow forregulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like).

Spacer Sequence

The spacer sequence hybridizes to a sequence in a target nucleic acid ofinterest. The spacer of a genome-targeting nucleic acid interacts with atarget nucleic acid in a sequence-specific manner via hybridization(i.e., base pairing). The nucleotide sequence of the spacer thus variesdepending on the sequence of the target nucleic acid of interest.

In a CRISPR/Cas system herein, the spacer sequence is designed tohybridize to a target nucleic acid that is located 5′ of a PAM of theCas9 enzyme used in the system. The spacer may perfectly match thetarget sequence or may have mismatches. Each Cas9 enzyme has aparticular PAM sequence that it recognizes in a target DNA. For example,S. pyogenes recognizes in a target nucleic acid a PAM that comprises thesequence 5′-NRG-3′, where R comprises either A or G, where N is anynucleotide and N is immediately 3′ of the target nucleic acid sequencetargeted by the spacer sequence.

In some embodiments, the target nucleic acid sequence comprises 20nucleotides. In some embodiments, the target nucleic acid comprises lessthan 20 nucleotides. In some embodiments, the target nucleic acidcomprises more than 20 nucleotides. In some embodiments, the targetnucleic acid comprises at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30 or more nucleotides. In some embodiments, the targetnucleic acid comprises at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30 or more nucleotides. In some embodiments, the targetnucleic acid sequence comprises 20 bases immediately 5′ of the firstnucleotide of the PAM. For example, in a sequence comprising5′-NNNNNNNNNNNNNNNNNNNNNRG-3′ (SEQ ID NO: 68,298), the target nucleicacid comprises the sequence that corresponds to the Ns, wherein N is anynucleotide, and the underlined NRG sequence is the S. pyogenes PAM.

In some embodiments, the spacer sequence that hybridizes to the targetnucleic acid has a length of at least about 6 nucleotides (nt). Thespacer sequence can be at least about 6 nt, at least about 10 nt, atleast about 15 nt, at least about 18 nt, at least about 19 nt, at leastabout 20 nt, at least about 25 nt, at least about 30 nt, at least about35 nt or at least about 40 nt, from about 6 nt to about 80 nt, fromabout 6 nt to about 50 nt, from about 6 nt to about 45 nt, from about 6nt to about 40 nt, from about 6 nt to about 35 nt, from about 6 nt toabout 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20nt, from about 6 nt to about 19 nt, from about 10 nt to about 50 nt,from about 10 nt to about 45 nt, from about 10 nt to about 40 nt, fromabout 10 nt to about 35 nt, from about 10 nt to about 30 nt, from about10 nt to about 25 nt, from about 10 nt to about 20 nt, from about 10 ntto about 19 nt, from about 19 nt to about 25 nt, from about 19 nt toabout 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt,from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, fromabout 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 ntto about 50 nt, or from about 20 nt to about 60 nt. In some embodiments,the spacer sequence comprises 20 nucleotides. In some embodiments, thespacer comprises 19 nucleotides.

In some embodiments, the percent complementarity between the spacersequence and the target nucleic acid is at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or 100%. In some embodiments,the percent complementarity between the spacer sequence and the targetnucleic acid is at most about 30%, at most about 40%, at most about 50%,at most about 60%, at most about 65%, at most about 70%, at most about75%, at most about 80%, at most about 85%, at most about 90%, at mostabout 95%, at most about 97%, at most about 98%, at most about 99%, or100%. In some embodiments, the percent complementarity between thespacer sequence and the target nucleic acid is 100% over the sixcontiguous 5′-most nucleotides of the target sequence of thecomplementary strand of the target nucleic acid. In some embodiments,the percent complementarity between the spacer sequence and the targetnucleic acid is at least 60% over about 20 contiguous nucleotides. Thelength of the spacer sequence and the target nucleic acid can differ by1 to 6 nucleotides, which may be thought of as a bulge or bulges.

In some embodiments, a spacer sequence is designed or chosen using acomputer program. The computer program can use variables, such aspredicted melting temperature, secondary structure formation, predictedannealing temperature, sequence identity, genomic context, chromatinaccessibility, % GC, frequency of genomic occurrence (e.g., of sequencesthat are identical or are similar but vary in one or more spots as aresult of mismatch, insertion or deletion), methylation status, presenceof SNPs, and the like.

Minimum CRISPR Repeat Sequence

In some embodiments, a minimum CRISPR repeat sequence is a sequence withat least about 30%, about 40%, about 50%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100%sequence identity to a reference CRISPR repeat sequence (e.g., crRNAfrom S. pyogenes).

A minimum CRISPR repeat sequence comprises nucleotides that canhybridize to a minimum tracrRNA sequence in a cell. The minimum CRISPRrepeat sequence and a minimum tracrRNA sequence form a duplex, i.e. abase-paired double-stranded structure. Together, the minimum CRISPRrepeat sequence and the minimum tracrRNA sequence bind to thesite-directed polypeptide. At least a part of the minimum CRISPR repeatsequence hybridizes to the minimum tracrRNA sequence. In someembodiments, at least a part of the minimum CRISPR repeat sequencecomprises at least about 30%, about 40%, about 50%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or 100% complementary to the minimum tracrRNA sequence. In someembodiments, at least a part of the minimum CRISPR repeat sequencecomprises at most about 30%, about 40%, about 50%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or100% complementary to the minimum tracrRNA sequence.

The minimum CRISPR repeat sequence can have a length from about 7nucleotides to about 100 nucleotides. For example, the length of theminimum CRISPR repeat sequence is from about 7 nucleotides (nt) to about50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt,from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, fromabout 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt toabout 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, orfrom about 15 nt to about 25 nt. In some embodiments, the minimum CRISPRrepeat sequence is approximately 9 nucleotides in length. In someembodiments, the minimum CRISPR repeat sequence is approximately 12nucleotides in length.

In some embodiments, the minimum CRISPR repeat sequence is at leastabout 60% identical to a reference minimum CRISPR repeat sequence (e.g.,wild-type crRNA from S. pyogenes) over a stretch of at least 6, 7, or 8contiguous nucleotides. For example, the minimum CRISPR repeat sequenceis at least about 65% identical, at least about 70% identical, at leastabout 75% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical, at least about 95% identical,at least about 98% identical, at least about 99% identical or 100%identical to a reference minimum CRISPR repeat sequence over a stretchof at least 6, 7, or 8 contiguous nucleotides.

Minimum tracrRNA Sequence

In some embodiments, a minimum tracrRNA sequence is a sequence with atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequenceidentity to a reference tracrRNA sequence (e.g., wild type tracrRNA fromS. pyogenes).

A minimum tracrRNA sequence comprises nucleotides that hybridize to aminimum CRISPR repeat sequence in a cell. A minimum tracrRNA sequenceand a minimum CRISPR repeat sequence form a duplex, i.e. a base-paireddouble-stranded structure. Together, the minimum tracrRNA sequence andthe minimum CRISPR repeat bind to a site-directed polypeptide. At leasta part of the minimum tracrRNA sequence can hybridize to the minimumCRISPR repeat sequence. In some embodiments, the minimum tracrRNAsequence is at least about 30%, about 40%, about 50%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or 100% complementary to the minimum CRISPR repeat sequence.

The minimum tracrRNA sequence can have a length from about 7 nucleotidesto about 100 nucleotides. For example, the minimum tracrRNA sequence canbe from about 7 nucleotides (nt) to about 50 nt, from about 7 nt toabout 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt long.In some embodiments, the minimum tracrRNA sequence is approximately 9nucleotides in length. In some embodiments, the minimum tracrRNAsequence is approximately 12 nucleotides. In some embodiments, theminimum tracrRNA consists of tracrRNA nt 23-48 described in Jinek etal., supra.

In some embodiments, the minimum tracrRNA sequence is at least about 60%identical to a reference minimum tracrRNA (e.g., wild type, tracrRNAfrom S. pyogenes) sequence over a stretch of at least 6, 7, or 8contiguous nucleotides. For example, the minimum tracrRNA sequence is atleast about 65% identical, about 70% identical, about 75% identical,about 80% identical, about 85% identical, about 90% identical, about 95%identical, about 98% identical, about 99% identical or 100% identical toa reference minimum tracrRNA sequence over a stretch of at least 6, 7,or 8 contiguous nucleotides.

In some embodiments, the duplex between the minimum CRISPR RNA and theminimum tracrRNA comprises a double helix. In some embodiments, theduplex between the minimum CRISPR RNA and the minimum tracrRNA comprisesat least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides. Insome embodiments, the duplex between the minimum CRISPR RNA and theminimum tracrRNA comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 or more nucleotides.

In some embodiments, the duplex comprises a mismatch (i.e., the twostrands of the duplex are not 100% complementary). In some embodiments,the duplex comprises at least about 1, 2, 3, 4, or 5 or mismatches. Insome embodiments, the duplex comprises at most about 1, 2, 3, 4, or 5 ormismatches. In some embodiments, the duplex comprises no more than 2mismatches.

Bulges

In some embodiments, there is a “bulge” in the duplex between theminimum CRISPR RNA and the minimum tracrRNA. The bulge is an unpairedregion of nucleotides within the duplex. In some embodiments, the bulgecontributes to the binding of the duplex to the site-directedpolypeptide. A bulge comprises, on one side of the duplex, an unpaired5′-XXXY-3′ where X is any purine and Y comprises a nucleotide that canform a wobble pair with a nucleotide on the opposite strand, and anunpaired nucleotide region on the other side of the duplex. The numberof unpaired nucleotides on the two sides of the duplex can be different.

In one example, the bulge comprises an unpaired purine (e.g., adenine)on the minimum CRISPR repeat strand of the bulge. In some embodiments, abulge comprises an unpaired 5′-AAGY-3′ of the minimum tracrRNA sequencestrand of the bulge, where Y comprises a nucleotide that can form awobble pairing with a nucleotide on the minimum CRISPR repeat strand.

In some embodiments, a bulge on the minimum CRISPR repeat side of theduplex comprises at least 1, 2, 3, 4, or 5 or more unpaired nucleotides.In some embodiments, a bulge on the minimum CRISPR repeat side of theduplex comprises at most 1, 2, 3, 4, or 5 or more unpaired nucleotides.In some embodiments, a bulge on the minimum CRISPR repeat side of theduplex comprises 1 unpaired nucleotide.

In some embodiments, a bulge on the minimum tracrRNA sequence side ofthe duplex comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or moreunpaired nucleotides. In some embodiments, a bulge on the minimumtracrRNA sequence side of the duplex comprises at most 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 or more unpaired nucleotides. In some embodiments, abulge on a second side of the duplex (e.g., the minimum tracrRNAsequence side of the duplex) comprises 4 unpaired nucleotides.

In some embodiments, a bulge comprises at least one wobble pairing. Insome embodiments, a bulge comprises at most one wobble pairing. In someembodiments, a bulge comprises at least one purine nucleotide. In someembodiments, a bulge comprises at least 3 purine nucleotides. In someembodiments, a bulge sequence comprises at least 5 purine nucleotides.In some embodiments, a bulge sequence comprises at least one guaninenucleotide. In some embodiments, a bulge sequence comprises at least oneadenine nucleotide.

Hairpins

In various embodiments, one or more hairpins are located 3′ to theminimum tracrRNA in the 3′ tracrRNA sequence.

In some embodiments, the hairpin starts at least about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, or 20 or more nucleotides 3′ from the last pairednucleotide in the minimum CRISPR repeat and minimum tracrRNA sequenceduplex. In some embodiments, the hairpin can start at most about 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides 3′ of the last pairednucleotide in the minimum CRISPR repeat and minimum tracrRNA sequenceduplex.

In some embodiments, a hairpin comprises at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, or 20 or more consecutive nucleotides. In someembodiments, a hairpin comprises at most about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, or more consecutive nucleotides.

In some embodiments, a hairpin comprises a CC dinucleotide (i.e., twoconsecutive cytosine nucleotides).

In some embodiments, a hairpin comprises duplexed nucleotides (e.g.,nucleotides in a hairpin, hybridized together). For example, a hairpincomprises a CC dinucleotide that is hybridized to a GG dinucleotide in ahairpin duplex of the 3′ tracrRNA sequence.

One or more of the hairpins can interact with guide RNA-interactingregions of a site-directed polypeptide.

In some embodiments, there are two or more hairpins, and in someembodiments there are three or more hairpins.

3′ tracrRNA Sequence

In some embodiments, a 3′ tracrRNA sequence comprises a sequence with atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequenceidentity to a reference tracrRNA sequence (e.g., a tracrRNA from S.pyogenes).

In some embodiments, the 3′ tracrRNA sequence has a length from about 6nucleotides to about 100 nucleotides. For example, the 3′ tracrRNAsequence can have a length from about 6 nucleotides (nt) to about 50 nt,from about 6 nt to about 40 nt, from about 6 nt to about 30 nt, fromabout 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt toabout 30 nt, from about 8 nt to about 25 nt, from about 8 nt to about 20nt, from about 8 nt to about 15 nt, from about 15 nt to about 100 nt,from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, fromabout 15 nt to about 40 nt, from about 15 nt to about 30 nt, or fromabout 15 nt to about 25 nt. In some embodiments, the 3′ tracrRNAsequence has a length of approximately 14 nucleotides.

In some embodiments, the 3′ tracrRNA sequence is at least about 60%identical to a reference 3′ tracrRNA sequence (e.g., wild type 3′tracrRNA sequence from S. pyogenes) over a stretch of at least 6, 7, or8 contiguous nucleotides. For example, the 3′ tracrRNA sequence is atleast about 60% identical, about 65% identical, about 70% identical,about 75% identical, about 80% identical, about 85% identical, about 90%identical, about 95% identical, about 98% identical, about 99%identical, or 100% identical, to a reference 3′ tracrRNA sequence (e.g.,wild type 3′ tracrRNA sequence from S. pyogenes) over a stretch of atleast 6, 7, or 8 contiguous nucleotides.

In some embodiments, a 3′ tracrRNA sequence comprises more than oneduplexed region (e.g., hairpin, hybridized region). In some embodiments,a 3′ tracrRNA sequence comprises two duplexed regions.

In some embodiments, the 3′ tracrRNA sequence comprises a stem loopstructure. In some embodiments, a stem loop structure in the 3′ tracrRNAcomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 or morenucleotides. In some embodiments, the stem loop structure in the 3′tracrRNA comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or morenucleotides. In some embodiments, the stem loop structure comprises afunctional moiety. For example, the stem loop structure may comprise anaptamer, a ribozyme, a protein-interacting hairpin, a CRISPR array, anintron, or an exon. In some embodiments, the stem loop structurecomprises at least about 1, 2, 3, 4, or 5 or more functional moieties.In some embodiments, the stem loop structure comprises at most about 1,2, 3, 4, or 5 or more functional moieties.

In some embodiments, the hairpin in the 3′ tracrRNA sequence comprises aP-domain. In some embodiments, the P-domain comprises a double-strandedregion in the hairpin.

tracrRNA Extension Sequence

A tracrRNA extension sequence may be provided whether the tracrRNA is inthe context of single-molecule guides or double-molecule guides. In someembodiments, a tracrRNA extension sequence has a length from about 1nucleotide to about 400 nucleotides. In some embodiments, a tracrRNAextension sequence has a length of more than 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240,260, 280, 300, 320, 340, 360, 380, or 400 nucleotides. In someembodiments, a tracrRNA extension sequence has a length from about 20 toabout 5000 or more nucleotides. In some embodiments, a tracrRNAextension sequence has a length of more than 1000 nucleotides. In someembodiments, a tracrRNA extension sequence has a length of less than 1,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140,160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or morenucleotides. In some embodiments, a tracrRNA extension sequence can havea length of less than 1000 nucleotides. In some embodiments, a tracrRNAextension sequence comprises less than 10 nucleotides in length. In someembodiments, a tracrRNA extension sequence is 10-30 nucleotides inlength. In some embodiments, tracrRNA extension sequence is 30-70nucleotides in length.

In some embodiments, the tracrRNA extension sequence comprises afunctional moiety (e.g., a stability control sequence, ribozyme,endoribonuclease binding sequence). In some embodiments, the functionalmoiety comprises a transcriptional terminator segment (i.e., atranscription termination sequence). In some embodiments, the functionalmoiety has a total length from about 10 nucleotides (nt) to about 100nucleotides, from about 10 nt to about 20 nt, from about 20 nt to about30 nt, from about 30 nt to about 40 nt, from about 40 nt to about 50 nt,from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, fromabout 70 nt to about 80 nt, from about 80 nt to about 90 nt, or fromabout 90 nt to about 100 nt, from about 15 nt to about 80 nt, from about15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 ntto about 30 nt, or from about 15 nt to about 25 nt. In some embodiments,the functional moiety functions in a eukaryotic cell. In someembodiments, the functional moiety functions in a prokaryotic cell. Insome embodiments, the functional moiety functions in both eukaryotic andprokaryotic cells.

Non-limiting examples of suitable tracrRNA extension functional moietiesinclude a 3′ poly-adenylated tail, a riboswitch sequence (e.g., to allowfor regulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like). In some embodiments, a tracrRNAextension sequence comprises a primer binding site or a molecular index(e.g., barcode sequence). In some embodiments, the tracrRNA extensionsequence comprises one or more affinity tags.

Single-Molecule Guide Linker Sequence

In some embodiments, the linker sequence of a single-molecule guidenucleic acid has a length from about 3 nucleotides to about 100nucleotides. In Jinek et al., supra, for example, a simple 4 nucleotide“tetraloop” (-GAAA-) was used, Science, 337(6096):816-821 (2012). Anillustrative linker has a length from about 3 nucleotides (nt) to about90 nt, from about 3 nt to about 80 nt, from about 3 nt to about 70 nt,from about 3 nt to about 60 nt, from about 3 nt to about 50 nt, fromabout 3 nt to about 40 nt, from about 3 nt to about 30 nt, from about 3nt to about 20 nt, from about 3 nt to about 10 nt. For example, thelinker can have a length from about 3 nt to about 5 nt, from about 5 ntto about 10 nt, from about 10 nt to about 15 nt, from about 15 nt toabout 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt,from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, fromabout 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about80 nt to about 90 nt, or from about 90 nt to about 100 nt. In someembodiments, the linker of a single-molecule guide nucleic acid isbetween 4 and 40 nucleotides. In some embodiments, a linker is at leastabout 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000,5500, 6000, 6500, or 7000 or more nucleotides. In some embodiments, alinker is at most about 100, 500, 1000, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000, 5500, 6000, 6500, or 7000 or more nucleotides.

Linkers can comprise any of a variety of sequences, although preferablythe linker will not comprise sequences that have extensive regions ofhomology with other portions of the guide RNA, which might causeintramolecular binding that could interfere with other functionalregions of the guide. In Jinek et al., supra, a simple 4 nucleotidesequence -GAAA- was used, Science, 337(6096):816-821 (2012), butnumerous other sequences, including longer sequences can likewise beused.

In some embodiments, the linker sequence comprises a functional moiety.For example, the linker sequence may comprise one or more features,including an aptamer, a ribozyme, a protein-interacting hairpin, aprotein binding site, a CRISPR array, an intron, or an exon. In someembodiments, the linker sequence comprises at least about 1, 2, 3, 4, or5 or more functional moieties. In some embodiments, the linker sequencecomprises at most about 1, 2, 3, 4, or 5 or more functional moieties.

Genome Engineering Strategies to Correct Cells by Insertion, Correction,Deletion, or Replacement of One or More Mutations or Exons within orNear the Gene, or by Knocking-in SERPINA1 cDNA or a Minigene into theLocus of the Corresponding Gene or Safe Harbor Site

The methods of the present disclosure can involve correction of one orboth of the mutant alleles. Gene editing to correct the mutation has theadvantage of restoration of correct expression levels and temporalcontrol. Sequencing the patient's SERPINA1 alleles allows for design ofthe gene editing strategy to best correct the identified mutation(s).

A step of the ex vivo methods of the invention involvesediting/correcting the patient specific iPS cells using genomeengineering. Alternatively, a step of the ex vivo methods of theinvention involves editing/correcting the progenitor cell, primaryhepatocyte, mesenchymal stem cell, or liver progenitor cell. Likewise, astep of the in vivo methods of the invention involves editing/correctingthe cells in an AATD patient using genome engineering. Similarly, a stepin the cellular methods of the invention involves editing/correcting theSERPINA1 gene in a human cell by genome engineering.

AATD patients exhibit one or more mutations in the SERPINA1 gene.Therefore, different patients will generally require differentcorrection strategies. Any CRISPR endonuclease may be used in themethods of the invention, each CRISPR endonuclease having its ownassociated PAM, which may or may not be disease specific. For example,gRNA spacer sequences for targeting the SERPINA1 gene with a CRISPR/Cas9endonuclease from S. pyogenes have been identified in SEQ ID NOs:54,860-61,324. gRNA spacer sequences for targeting the SERPINA1 genewith a CRISPR/Cas9 endonuclease from S. aureus have been identified inSEQ ID NOs: 61,325-61,936. gRNA spacer sequences for targeting theSERPINA1 gene with a CRISPR/Cas9 endonuclease from S. thermophilus havebeen identified in SEQ ID NOs: 61,9347-62,069. gRNA spacer sequences fortargeting the SERPINA1 gene with a CRISPR/Cas9 endonuclease from Tdenticola have been identified in SEQ ID NOs: 62,070-62,120. gRNA spacersequences for targeting the SERPINA1 gene with a CRISPR/Cas9endonuclease from N. meningitides have been identified in SEQ ID NOs:62,121-62,563. gRNA spacer sequences for targeting the SERPINA1 genewith a CRISPR/Cpf1 endonuclease from Acidominococcus, Lachnospiraceae,and Francisella novicida have been identified in SEQ ID NOs:62,564-68,297. gRNA spacer sequences for targeting, e.g., targeting exon1-2 of AAVS1 (PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX (F9),G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, and TTR with a CRISPR/Cas9endonuclease from S. pyogenes have been identified in Example 7. gRNAspacer sequences for targeting, e.g., targeting exon 1-2 of, AAVS1(PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD,Lp(a), Pcsk9, Serpina1, TF, and TTR with a CRISPR/Cas9 endonuclease fromS. aureus have been identified in Example 8. gRNA spacer sequences fortargeting, e.g., targeting exon 1-2 of, AAVS1 (PPP1R12C), ALB, Angptl3,ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1,TF, and TTR with a CRISPR/Cas9 endonuclease from S. thermophilus havebeen identified in Example 9. gRNA spacer sequences for targeting, e.g.,targeting exon 1-2 of, AAVS1 (PPP1R12C), ALB, Angptl3, ApoC3, ASGR2,CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, and TTRwith a CRISPR/Cas9 endonuclease from T. denticola have been identifiedin Example 10. gRNA spacer sequences for targeting, e.g., targeting exon1-2 of, AAVS1 (PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX (F9),G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, and TTR with a CRISPR/Cas9endonuclease from N. meningitides have been identified in Example 11.gRNA spacer sequences for targeting, e.g., targeting exon 1-2 of, AAVS1(PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD,Lp(a), Pcsk9, Serpina1, TF, and TTR with a CRISPR/Cas9 endonuclease fromAcidominococcus, Lachnospiraceae, and Francisella novicida have beenidentified in Example 12.

For example, the mutation can be corrected by the insertions ordeletions that arise due to the imprecise NHEJ repair pathway. If thepatient's SERPINA1 gene has an inserted or deleted base, a targetedcleavage can result in a NHEJ-mediated insertion or deletion thatrestores the frame. Missense mutations can also be corrected throughNHEJ-mediated correction using one or more guide RNA. The ability orlikelihood of the cut(s) to correct the mutation can be designed orevaluated based on the local sequence and micro-homologies. NHEJ canalso be used to delete segments of the gene, either directly or byaltering splice donor or acceptor sites through cleavage by one gRNAtargeting several locations, or several gRNAs. This may be useful if anamino acid, domain or exon contains the mutations and can be removed orinverted, or if the deletion otherwise restored function to the protein.Pairs of guide strands have been used for deletions and corrections ofinversions.

Alternatively, the donor for correction by HDR contains the correctedsequence with small or large flanking homology arms to allow forannealing. HDR is essentially an error-free mechanism that uses asupplied homologous DNA sequence as a template during DSB repair. Therate of homology directed repair (HDR) is a function of the distancebetween the mutation and the cut site so choosing overlapping or nearesttarget sites is important. Templates can include extra sequences flankedby the homologous regions or can contain a sequence that differs fromthe genomic sequence, thus allowing sequence editing.

In addition to correcting mutations by NHEJ or HDR, a range of otheroptions are possible. If there are small or large deletions or multiplemutations, a cDNA can be knocked in that contains the exons affected. Afull length cDNA can be knocked into any “safe harbor”, but must use asupplied or other promoter. If this construct is knocked into thecorrect location, it will have physiological control, similar to thenormal gene. Pairs of nucleases can be used to delete mutated generegions, though a donor would usually have to be provided to restorefunction. In this case two gRNA would be supplied and one donorsequence.

Some genome engineering strategies involve correction of one or moremutations in or near the SERPINA1 gene or deleting the mutant SERPINA1DNA and/or knocking-in SERPINA1 cDNA or a minigene (comprised of one ormore exons and introns or natural or synthetic introns) into the locusof the corresponding gene or a safe harbor locus by homology directedrepair (HDR), which is also known as homologous recombination (HR).Homology directed repair is one strategy for treating patients that haveinactivating mutations in or near the SERPINA1 gene. These strategieswill restore the SERPINA1 gene and completely reverse, treat, and/ormitigate the diseased state. This strategy will require a more customapproach based on the location of the patient's inactivatingmutation(s). Donor nucleotides for correcting mutations are small (<300bp). This is advantageous, as HDR efficiencies may be inversely relatedto the size of the donor molecule. Also, it is expected that the donortemplates can fit into size constrained viral vector molecules, e.g.,adeno-associated virus (AAV) molecules, which have been shown to be aneffective means of donor template delivery. Also, it is expected thatthe donor templates can fit into other size constrained molecules,including, by way of non-limiting example, platelets and/or exosomes orother microvesicles.

Homology direct repair is a cellular mechanism for repairingdouble-stranded breaks (DSBs). The most common form is homologousrecombination. There are additional pathways for HDR, includingsingle-strand annealing and alternative-HDR. Genome engineering toolsallow researchers to manipulate the cellular homologous recombinationpathways to create site-specific modifications to the genome. It hasbeen found that cells can repair a double-stranded break using asynthetic donor molecule provided in trans. Therefore, by introducing adouble-stranded break near a specific mutation and providing a suitabledonor, targeted changes can be made in the genome. Specific cleavageincreases the rate of HDR more than 1,000 fold above the rate of 1 in10⁵ cells receiving a homologous donor alone. The rate of homologydirected repair (HDR) at a particular nucleotide is a function of thedistance to the cut site, so choosing overlapping or nearest targetsites is important. Gene editing offers the advantage over geneaddition, as correcting in situ leaves the rest of the genomeunperturbed.

Supplied donors for editing by HDR vary markedly but generally containthe intended sequence with small or large flanking homology arms toallow annealing to the genomic DNA. The homology regions flanking theintroduced genetic changes can be 30 bp or smaller, or as large as amulti-kilobase cassette that can contain promoters, cDNAs, etc. Bothsingle-stranded and double-stranded oligonucleotide donors have beenused. These oligonucleotides range in size from less than 100 nt to overmany kb, though longer ssDNA can also be generated and used.Double-stranded donors are often used, including PCR amplicons,plasmids, and mini-circles. In general, it has been found that an AAVvector is a very effective means of delivery of a donor template, thoughthe packaging limits for individual donors is <5 kb. Activetranscription of the donor increased HDR three-fold, indicating theinclusion of promoter may increase conversion. Conversely, CpGmethylation of the donor decreased gene expression and HDR.

In addition to wildtype endonucleases, such as Cas9, nickase variantsexist that have one or the other nuclease domain inactivated resultingin cutting of only one DNA strand. HDR can be directed from individualCas nickases or using pairs of nickases that flank the target area.Donors can be single-stranded, nicked, or dsDNA.

The donor DNA can be supplied with the nuclease or independently by avariety of different methods, for example by transfection,nano-particle, micro-injection, or viral transduction. A range oftethering options has been proposed to increase the availability of thedonors for HDR. Examples include attaching the donor to the nuclease,attaching to DNA binding proteins that bind nearby, or attaching toproteins that are involved in DNA end binding or repair.

The repair pathway choice can be guided by a number of cultureconditions, such as those that influence cell cycling, or by targetingof DNA repair and associated proteins. For example, to increase HDR, keyNHEJ molecules can be suppressed, such as KU70, KU80 or DNA ligase IV.

Without a donor present, the ends from a DNA break or ends fromdifferent breaks can be joined using the several nonhomologous repairpathways in which the DNA ends are joined with little or no base-pairingat the junction. In addition to canonical NHEJ, there are similar repairmechanisms, such as alt-NHEJ. If there are two breaks, the interveningsegment can be deleted or inverted. NHEJ repair pathways can lead toinsertions, deletions or mutations at the joints.

NHEJ was used to insert a 15-kb inducible gene expression cassette intoa defined locus in human cell lines after nuclease cleavage. Maresca,M., Lin, V. G., Guo, N. & Yang, Y., Genome Res 23, 539-546 (2013).

In addition to genome editing by NHEJ or HDR, site-specific geneinsertions have been conducted that use both the NHEJ pathway and HR. Acombination approach may be applicable in certain settings, possiblyincluding intron/exon borders. NHEJ may prove effective for ligation inthe intron, while the error-free HDR may be better suited in the codingregion.

As stated previously, the SERPINA1 gene contains 5 exons. Any one ormore of the 5 exons or nearby introns may be repaired in order tocorrect a mutation and restore the inactive AAT protein. Alternatively,there are various mutations associated with AATD, which are acombination of missense, nonsense, frameshift and other mutations, withthe common effect of inactivating AAT. Any one or more of the mutationsmay be repaired in order to restore the inactive AAT. For example, oneor more of the following pathological variants may be corrected:rs764325655, rs121912713, rs28929474, rs17580, rs121912714, rs764220898,rs199422211, rs751235320, rs199422210, rs267606950, rs55819880,rs28931570 (See Table 1). These variants include deletions, insertionsand single nucleotide polymorphisms. As a further alternative, SERPINA1cDNA or minigene (comprised of, natural or synthetic enhancer andpromoter, one or more exons, and natural or synthetic introns, andnatural or synthetic 3′UTR and polyadenylation signal) may be knocked-into the locus of the corresponding gene or knocked-in to a safe harborsite, such as AAVS1 (PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX(F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, and/or TTR. The safeharbor locus can be selected from the group consisting of: exon 1-2 ofAAVS1 (PPP1R12C), exon 1-2 of ALB, exon 1-2 of Angptl3, exon 1-2 ofApoC3, exon 1-2 of ASGR2, exon 1-2 of CCR5, exon 1-2 of FIX (F9), exon1-2 of G6PC, exon 1-2 of Gys2, exon 1-2 of HGD, exon 1-2 of Lp(a), exon1-2 of Pcsk9, exon 1-2 of Serpina1, exon 1-2 of TF, and exon 1-2 of TTR.In some embodiments, the methods provide one gRNA or a pair of gRNAsthat can be used to facilitate incorporation of a new sequence from apolynucleotide donor template to correct one or more mutations or toknock-in a part of or the entire SERPINA1 gene or cDNA.

TABLE 1 Variant Location Variant type rs764325655 94,378,547-94,378,548Insertion rs121912713 94,378,561 single nucleotide variation rs2892947494,378,610 single nucleotide variation rs17580 94,380,925 singlenucleotide variation rs121912714 94,380,949 single nucleotide variationrs764220898 94,381,043 single nucleotide variation rs19942221194,381,067 single nucleotide variation rs751235320 94,382,591 singlenucleotide variation rs199422210 94,382,686 single nucleotide variationrs267606950 94,382,686 Insertion rs55819880 94,383,008 single nucleotidevariation rs28931570 94,383,051 single nucleotide variation

Some embodiments of the methods provide gRNA pairs that make a deletionby cutting the gene twice, one gRNA cutting at the 5′ end of one or moremutations and the other gRNA cutting at the 3′ end of one or moremutations that facilitates insertion of a new sequence from apolynucleotide donor template to replace the one or more mutations, ordeletion may exclude mutant amino acids or amino acids adjacent to it(e.g., premature stop codon) and lead to expression of a functionalprotein, or restore an open reading frame. The cutting may beaccomplished by a pair of DNA endonucleases that each makes a DSB in thegenome, or by multiple nickases that together make a DSB in the genome.

Alternatively, some embodiments of the methods provide one gRNA to makeone double-strand cut around one or more mutations that facilitatesinsertion of a new sequence from a polynucleotide donor template toreplace the one or more mutations. The double-strand cut may be made bya single DNA endonuclease or multiple nickases that together make a DSBin the genome, or single gRNA may lead to deletion (MMEJ), which mayexclude mutant amino acid (e.g., premature stop codon) and lead toexpression of a functional protein, or restore an open reading frame.

Illustrative modifications within the SERPINA1 gene include replacementswithin or near (proximal) to the mutations referred to above, such aswithin the region of less than 3 kb, less than 2 kb, less than 1 kb,less than 0.5 kb upstream or downstream of the specific mutation. Giventhe relatively wide variations of mutations in the SERPINA1 gene, itwill be appreciated that numerous variations of the replacementsreferenced above (including without limitation larger as well as smallerdeletions), would be expected to result in restoration of AAT proteinactivity.

Such variants include replacements that are larger in the 5′ and/or 3′direction than the specific mutation in question, or smaller in eitherdirection. Accordingly, by “near” or “proximal” with respect to specificreplacements, it is intended that the SSB or DSB locus associated with adesired replacement boundary (also referred to herein as an endpoint)may be within a region that is less than about 3 kb from the referencelocus noted. In some embodiments, the DSB locus is more proximal andwithin 2 kb, within 1 kb, within 0.5 kb, or within 0.1 kb. In the caseof small replacement, the desired endpoint is at or “adjacent to” thereference locus, by which it is intended that the endpoint is within 100bp, within 50 bp, within 25 bp, or less than about 10 bp to 5 bp fromthe reference locus.

Embodiments comprising larger or smaller replacements are expected toprovide the same benefit, as long as the AAT protein activity isrestored. It is thus expected that many variations of the replacementsdescribed and illustrated herein will be effective for amelioratingAATD.

Another genome engineering strategy involves exon deletion. Targeteddeletion of specific exons is an attractive strategy for treating alarge subset of patients with a single therapeutic cocktail. Deletionscan either be single exon deletions or multi-exon deletions. Whilemulti-exon deletions can reach a larger number of patients, for largerdeletions the efficiency of deletion greatly decreases with increasedsize. Therefore, deletions range can be from 40 to 10,000 base pairs(bp) in size. For example, deletions may range from 40-100; 100-300;300-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; or5,000-10,000 base pairs in size.

Deletions can occur in enhancer, promoter, 1st intron, and/or 3′UTRleading to upregulation of the gene expression, and/or through deletionof the regulatory elements.

As stated previously, the AAT gene contains 5 exons. Any one or more ofthe 5 exons, or aberrant intronic splice acceptor or donor sites, may bedeleted in order to restore the AAT reading frame. In some embodiments,the methods provide gRNA pairs that can be used to delete exons 1, 2, 3,4, or 5, or any combination of them.

In order to ensure that the pre-mRNA is properly processed followingexon deletion, the surrounding splicing signals can be deleted. Splicingdonor and acceptors are generally within 100 base pairs of theneighboring intron. Therefore, in some examples, methods can provide allgRNAs that cut approximately +/−100-3100 bp with respect to eachexon/intron junction of interest.

For any of the genome editing strategies, gene editing can be confirmedby sequencing or PCR analysis.

Target Sequence Selection

Shifts in the location of the 5′ boundary and/or the 3′ boundaryrelative to particular reference loci are used to facilitate or enhanceparticular applications of gene editing, which depend in part on theendonuclease system selected for the editing, as further described andillustrated herein.

In a first, non-limiting example of such target sequence selection, manyendonuclease systems have rules or criteria that guide the initialselection of potential target sites for cleavage, such as therequirement of a PAM sequence motif in a particular position adjacent tothe DNA cleavage sites in the case of CRISPR Type II or Type Vendonucleases.

In another non-limiting example of target sequence selection oroptimization, the frequency of “off-target” activity for a particularcombination of target sequence and gene editing endonuclease (i.e. thefrequency of DSBs occurring at sites other than the selected targetsequence) is assessed relative to the frequency of on-target activity.In some cases, cells that have been correctly edited at the desiredlocus may have a selective advantage relative to other cells.Illustrative, but nonlimiting, examples of a selective advantage includethe acquisition of attributes such as enhanced rates of replication,persistence, resistance to certain conditions, enhanced rates ofsuccessful engraftment or persistence in vivo following introductioninto a patient, and other attributes associated with the maintenance orincreased numbers or viability of such cells. In other cases, cells thathave been correctly edited at the desired locus may be positivelyselected for by one or more screening methods used to identify, sort orotherwise select for cells that have been correctly edited. Bothselective advantage and directed selection methods may take advantage ofthe phenotype associated with the correction. In some embodiments, cellsmay be edited two or more times in order to create a second modificationthat creates a new phenotype that is used to select or purify theintended population of cells. Such a second modification could becreated by adding a second gRNA for a selectable or screenable marker.In some cases, cells can be correctly edited at the desired locus usinga DNA fragment that contains the cDNA and also a selectable marker.

Whether any selective advantage is applicable or any directed selectionis to be applied in a particular case, target sequence selection is alsoguided by consideration of off-target frequencies in order to enhancethe effectiveness of the application and/or reduce the potential forundesired alterations at sites other than the desired target. Asdescribed further and illustrated herein and in the art, the occurrenceof off-target activity is influenced by a number of factors includingsimilarities and dissimilarities between the target site and various offtarget sites, as well as the particular endonuclease used.Bioinformatics tools are available that assist in the prediction ofoff-target activity, and frequently such tools can also be used toidentify the most likely sites of off-target activity, which can then beassessed in experimental settings to evaluate relative frequencies ofoff-target to on-target activity, thereby allowing the selection ofsequences that have higher relative on-target activities. Illustrativeexamples of such techniques are provided herein, and others are known inthe art.

Another aspect of target sequence selection relates to homologousrecombination events. Sequences sharing regions of homology can serve asfocal points for homologous recombination events that result in deletionof intervening sequences. Such recombination events occur during thenormal course of replication of chromosomes and other DNA sequences, andalso at other times when DNA sequences are being synthesized, such as inthe case of repairs of double-strand breaks (DSBs), which occur on aregular basis during the normal cell replication cycle but may also beenhanced by the occurrence of various events (such as UV light and otherinducers of DNA breakage) or the presence of certain agents (such asvarious chemical inducers). Many such inducers cause DSBs to occurindiscriminately in the genome, and DSBs are regularly being induced andrepaired in normal cells. During repair, the original sequence may bereconstructed with complete fidelity, however, in some cases, smallinsertions or deletions (referred to as “indels”) are introduced at theDSB site.

DSBs may also be specifically induced at particular locations, as in thecase of the endonucleases systems described herein, which can be used tocause directed or preferential gene modification events at selectedchromosomal locations. The tendency for homologous sequences to besubject to recombination in the context of DNA repair (as well asreplication) can be taken advantage of in a number of circumstances, andis the basis for one application of gene editing systems, such asCRISPR, in which homology directed repair is used to insert a sequenceof interest, provided through use of a “donor” polynucleotide, into adesired chromosomal location.

Regions of homology between particular sequences, which can be smallregions of “microhomology” that may comprise as few as ten basepairs orless, can also be used to bring about desired deletions. For example, asingle DSB is introduced at a site that exhibits microhomology with anearby sequence. During the normal course of repair of such DSB, aresult that occurs with high frequency is the deletion of theintervening sequence as a result of recombination being facilitated bythe DSB and concomitant cellular repair process.

In some circumstances, however, selecting target sequences withinregions of homology can also give rise to much larger deletions,including gene fusions (when the deletions are in coding regions), whichmay or may not be desired given the particular circumstances.

The examples provided herein further illustrate the selection of varioustarget regions for the creation of DSBs designed to induce replacementsthat result in restoration of AAT protein activity, as well as theselection of specific target sequences within such regions that aredesigned to minimize off-target events relative to on-target events.

Nucleic Acid Modifications

In some embodiments, polynucleotides introduced into cells comprise oneor more modifications that can be used individually or in combination,for example, to enhance activity, stability or specificity, alterdelivery, reduce innate immune responses in host cells, or for otherenhancements, as further described herein and known in the art.

In certain embodiments, modified polynucleotides are used in theCRISPR/Cas9/Cpf1 system, in which case the guide RNAs (eithersingle-molecule guides or double-molecule guides) and/or a DNA or an RNAencoding a Cas or Cpf1 endonuclease introduced into a cell can bemodified, as described and illustrated below. Such modifiedpolynucleotides can be used in the CRISPR/Cas9/Cpf1 system to edit anyone or more genomic loci.

Using the CRISPR/Cas9/Cpf1 system for purposes of nonlimitingillustrations of such uses, modifications of guide RNAs can be used toenhance the formation or stability of the CRISPR/Cas9/Cpf1 genomeediting complex comprising guide RNAs, which may be single-moleculeguides or double-molecule, and a Cas or Cpf1 endonuclease. Modificationsof guide RNAs can also or alternatively be used to enhance theinitiation, stability or kinetics of interactions between the genomeediting complex with the target sequence in the genome, which can beused, for example, to enhance on-target activity. Modifications of guideRNAs can also or alternatively be used to enhance specificity, e.g., therelative rates of genome editing at the on-target site as compared toeffects at other (off-target) sites.

Modifications can also or alternatively be used to increase thestability of a guide RNA, e.g., by increasing its resistance todegradation by ribonucleases (RNases) present in a cell, thereby causingits half-life in the cell to be increased. Modifications enhancing guideRNA half-life can be particularly useful in embodiments in which a Casor Cpf1 endonuclease is introduced into the cell to be edited via an RNAthat needs to be translated in order to generate endonuclease, becauseincreasing the half-life of guide RNAs introduced at the same time asthe RNA encoding the endonuclease can be used to increase the time thatthe guide RNAs and the encoded Cas or Cpf1 endonuclease co-exist in thecell.

Modifications can also or alternatively be used to decrease thelikelihood or degree to which RNAs introduced into cells elicit innateimmune responses. Such responses, which have been well characterized inthe context of RNA interference (RNAi), including small-interfering RNAs(siRNAs), as described below and in the art, tend to be associated withreduced half-life of the RNA and/or the elicitation of cytokines orother factors associated with immune responses.

One or more types of modifications can also be made to RNAs encoding anendonuclease that are introduced into a cell, including, withoutlimitation, modifications that enhance the stability of the RNA (such asby increasing its degradation by RNAses present in the cell),modifications that enhance translation of the resulting product (i.e.the endonuclease), and/or modifications that decrease the likelihood ordegree to which the RNAs introduced into cells elicit innate immuneresponses.

Combinations of modifications, such as the foregoing and others, canlikewise be used. In the case of CRISPR/Cas9/Cpf1, for example, one ormore types of modifications can be made to guide RNAs (including thoseexemplified above), and/or one or more types of modifications can bemade to RNAs encoding Cas endonuclease (including those exemplifiedabove).

By way of illustration, guide RNAs used in the CRISPR/Cas9/Cpf1 system,or other smaller RNAs can be readily synthesized by chemical means,enabling a number of modifications to be readily incorporated, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach used for generating chemically-modified RNAsof greater length is to produce two or more molecules that are ligatedtogether. Much longer RNAs, such as those encoding a Cas9 endonuclease,are more readily generated enzymatically. While fewer types ofmodifications are generally available for use in enzymatically producedRNAs, there are still modifications that can be used to, e.g., enhancestability, reduce the likelihood or degree of innate immune response,and/or enhance other attributes, as described further below and in theart; and new types of modifications are regularly being developed.

By way of illustration of various types of modifications, especiallythose used frequently with smaller chemically synthesized RNAs,modifications can comprise one or more nucleotides modified at the 2′position of the sugar, in some embodiments a 2′-O-alkyl,2′-O-alkyl-O-alkyl, or 2′-fluoro-modified nucleotide. In someembodiments, RNA modifications include 2′-fluoro, 2′-amino or 2′O-methyl modifications on the ribose of pyrimidines, abasic residues, oran inverted base at the 3′ end of the RNA. Such modifications areroutinely incorporated into oligonucleotides and these oligonucleotideshave been shown to have a higher Tm (i.e., higher target bindingaffinity) than 2′-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligonucleotide; these modifiedoligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Some oligonucleotides are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH2-NH—O—CH2, CH,˜N(CH3)˜O˜CH2 (known as amethylene(methylimino) or MMI backbone), CH2-O—N(CH3)-CH2,CH2-N(CH3)-N(CH3)-CH2 and O—N (CH3)-CH2-CH2 backbones, wherein thenative phosphodiester backbone is represented as O—P—O—CH,); amidebackbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)];morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.5,034,506); peptide nucleic acid (PNA) backbone (wherein thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone, see Nielsen et al.,Science 1991, 254, 1497). Phosphorus-containing linkages include, butare not limited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Braasch and DavidCorey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue3, (2001); Heasman, Dev. Biol., 243: 209-214 (2002); Nasevicius et al.,Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci.,97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH3, F, OCN, OCH3 OCH3,OCH3 O(CH2)n CH3, O(CH2)n NH2, or O(CH2)n CH3, where n is from 1 toabout 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O—, S—, or N-alkyl; O—, S—,or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. In some embodiments, amodification includes 2′-methoxyethoxy (2′-0-CH2CH2OCH3, also known as2′-O-(2-methoxyethyl)) (Martin et al, Helv. Chim. Acta, 1995, 78, 486).Other modifications include 2′-methoxy (2′-0-CH3), 2′-propoxy (2′-OCH2CH2CH3) and 2′-fluoro (2′-F). Similar modifications may also be made atother positions on the oligonucleotide, particularly the 3′ position ofthe sugar on the 3′ terminal nucleotide and the 5′ position of 5′terminal nucleotide. Oligonucleotides may also have sugar mimetics, suchas cyclobutyls in place of the pentofuranosyl group.

In some embodiments, both a sugar and an internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds comprise, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNAcompounds can be found in Nielsen et al, Science, 254: 1497-1500 (1991).

Guide RNAs can also include, additionally or alternatively, nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C), and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine, and2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co.,San Francisco, pp 75-77 (1980); Gebeyehu et al., Nucl. Acids Res.15:4513 (1997). A “universal” base known in the art, e.g., inosine, canalso be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke,S. T. and Lebleu, B., eds., Antisense Research and Applications, CRCPress, Boca Raton, 1993, pp. 276-278) and are embodiments of basesubstitutions.

Modified nucleobases comprise other synthetic and natural nucleobases,such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylquanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Theseinclude 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2C (Sanghvi, Y. S.,Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research andApplications’, CRC Press, Boca Raton, 1993, pp. 276-278) and areembodiments of base substitutions, even more particularly when combinedwith 2′-O-methoxyethyl sugar modifications. Modified nucleobases aredescribed in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588;5,830,653; 6,005,096; and U.S. Patent Application Publication2003/0158403.

Thus, the term “modified” refers to a non-natural sugar, phosphate, orbase that is incorporated into a guide RNA, an endonuclease, or both aguide RNA and an endonuclease. It is not necessary for all positions ina given oligonucleotide to be uniformly modified, and in fact more thanone of the aforementioned modifications may be incorporated in a singleoligonucleotide, or even in a single nucleoside within anoligonucleotide.

In some embodiments, the guide RNAs and/or mRNA (or DNA) encoding anendonuclease are chemically linked to one or more moieties or conjugatesthat enhance the activity, cellular distribution, or cellular uptake ofthe oligonucleotide. Such moieties comprise, but are not limited to,lipid moieties such as a cholesterol moiety [Letsinger et al., Proc.Natl. Acad. Sci. USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan etal., Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; a thioether, e.g.,hexyl-S-tritylthiol [Manoharan et al, Ann. N. Y. Acad. Sci., 660:306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let, 3:2765-2770 (1993)]; a thiocholesterol [Oberhauser et al., Nucl. AcidsRes., 20: 533-538 (1992)]; an aliphatic chain, e.g., dodecandiol orundecyl residues [Kabanov et al., FEBS Lett., 259: 327-330 (1990) andSvinarchuk et al., Biochimie, 75: 49-54 (1993)]; a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al.,Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. AcidsRes., 18: 3777-3783 (1990)]; a polyamine or a polyethylene glycol chain[Mancharan et al., Nucleosides & Nucleotides, 14: 969-973 (1995)];adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys.Acta, 1264: 229-237 (1995)]; or an octadecylamine orhexylamino-carbonyl-t oxycholesterol moiety [Crooke et al., J.Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See also U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941.

Sugars and other moieties can be used to target proteins and complexescomprising nucleotides, such as cationic polysomes and liposomes, toparticular sites. For example, hepatic cell directed transfer can bemediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, etal., Protein Pept Lett. 21(10):1025-30 (2014). Other systems known inthe art and regularly developed can be used to target biomolecules ofuse in the present case and/or complexes thereof to particular targetcells of interest.

These targeting moieties or conjugates can include conjugate groupscovalently bound to functional groups, such as primary or secondaryhydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23,1992, and U.S. Pat. No. 6,287,860, which are incorporated herein byreference. Conjugate moieties include, but are not limited to, lipidmoieties such as a cholesterol moiety, cholic acid, a thioether, e.g.,hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See,e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and5,688,941.

Longer polynucleotides that are less amenable to chemical synthesis andare typically produced by enzymatic synthesis can also be modified byvarious means. Such modifications can include, for example, theintroduction of certain nucleotide analogs, the incorporation ofparticular sequences or other moieties at the 5′ or 3′ ends ofmolecules, and other modifications. By way of illustration, the mRNAencoding Cas9 is approximately 4 kb in length and can be synthesized byin vitro transcription. Modifications to the mRNA can be applied to,e.g., increase its translation or stability (such as by increasing itsresistance to degradation with a cell), or to reduce the tendency of theRNA to elicit an innate immune response that is often observed in cellsfollowing introduction of exogenous RNAs, particularly longer RNAs suchas that encoding Cas9.

Numerous such modifications have been described in the art, such aspolyA tails, 5′ cap analogs (e.g., Anti Reverse Cap Analog (ARCA) orm7G(5′)ppp(5′)G (mCAP)), modified 5′ or 3′ untranslated regions (UTRs),use of modified bases (such as Pseudo-UTP, 2-Thio-UTP,5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP) or N6-Methyl-ATP), ortreatment with phosphatase to remove 5′ terminal phosphates. These andother modifications are known in the art, and new modifications of RNAsare regularly being developed.

There are numerous commercial suppliers of modified RNAs, including forexample, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon andmany others. As described by TriLink, for example, 5-Methyl-CTP can beused to impart desirable characteristics, such as increased nucleasestability, increased translation or reduced interaction of innate immunereceptors with in vitro transcribed RNA.5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP), N6-Methyl-ATP, as wellas Pseudo-UTP and 2-Thio-UTP, have also been shown to reduce innateimmune stimulation in culture and in vivo while enhancing translation,as illustrated in publications by Kormann et al. and Warren et al.referred to below.

It has been shown that chemically modified mRNA delivered in vivo can beused to achieve improved therapeutic effects; see, e.g., Kormann et al.,Nature Biotechnology 29, 154-157 (2011). Such modifications can be used,for example, to increase the stability of the RNA molecule and/or reduceits immunogenicity. Using chemical modifications such as Pseudo-U,N6-Methyl-A, 2-Thio-U and 5-Methyl-C, it was found that substitutingjust one quarter of the uridine and cytidine residues with 2-Thio-U and5-Methyl-C respectively resulted in a significant decrease in toll-likereceptor (TLR) mediated recognition of the mRNA in mice. By reducing theactivation of the innate immune system, these modifications can be usedto effectively increase the stability and longevity of the mRNA in vivo;see, e.g., Kormann et al., supra.

It has also been shown that repeated administration of syntheticmessenger RNAs incorporating modifications designed to bypass innateanti-viral responses can reprogram differentiated human cells topluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618-30(2010). Such modified mRNAs that act as primary reprogramming proteinscan be an efficient means of reprogramming multiple human cell types.Such cells are referred to as induced pluripotency stem cells (iPSCs),and it was found that enzymatically synthesized RNA incorporating5-Methyl-CTP, Pseudo-UTP and an Anti Reverse Cap Analog (ARCA) could beused to effectively evade the cell's antiviral response; see, e.g.,Warren et al., supra.

Other modifications of polynucleotides described in the art include, forexample, the use of polyA tails, the addition of 5′ cap analogs (such asm7G(5′)ppp(5′)G (mCAP)), modifications of 5′ or 3′ untranslated regions(UTRs), or treatment with phosphatase to remove 5′ terminalphosphates—and new approaches are regularly being developed.

A number of compositions and techniques applicable to the generation ofmodified RNAs for use herein have been developed in connection with themodification of RNA interference (RNAi), including small-interferingRNAs (siRNAs). siRNAs present particular challenges in vivo becausetheir effects on gene silencing via mRNA interference are generallytransient, which can require repeat administration. In addition, siRNAsare double-stranded RNAs (dsRNA) and mammalian cells have immuneresponses that have evolved to detect and neutralize dsRNA, which isoften a by-product of viral infection. Thus, there are mammalian enzymessuch as PKR (dsRNA-responsive kinase), and potentially retinoicacid-inducible gene I (RIG-I), that can mediate cellular responses todsRNA, as well as Toll-like receptors (such as TLR3, TLR7 and TLR8) thatcan trigger the induction of cytokines in response to such molecules;see, e.g., the reviews by Angart et al., Pharmaceuticals (Basel) 6(4):440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012);Burnett et al., Biotechnol J. 6(9):1130-46 (2011); Judge and MacLachlan,Hum Gene Ther 19(2):111-24 (2008); and references cited therein.

A large variety of modifications have been developed and applied toenhance RNA stability, reduce innate immune responses, and/or achieveother benefits that can be useful in connection with the introduction ofpolynucleotides into human cells, as described herein; see, e.g., thereviews by Whitehead K A et al., Annual Review of Chemical andBiomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, MiniRev Med Chem, 10(7):578-95 (2010); Chemolovskaya et al, Curr Opin MolTher., 12(2):158-67 (2010); Deleavey et al., Curr Protoc Nucleic AcidChem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19(2008); Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); Bremsenet al., Front Genet 3:154 (2012).

As noted above, there are a number of commercial suppliers of modifiedRNAs, many of which have specialized in modifications designed toimprove the effectiveness of siRNAs. A variety of approaches are offeredbased on various findings reported in the literature. For example,Dharmacon notes that replacement of a non-bridging oxygen with sulfur(phosphorothioate, PS) has been extensively used to improve nucleaseresistance of siRNAs, as reported by Kole, Nature Reviews Drug Discovery11:125-140 (2012). Modifications of the 2′-position of the ribose havebeen reported to improve nuclease resistance of the internucleotidephosphate bond while increasing duplex stability (Tm), which has alsobeen shown to provide protection from immune activation. A combinationof moderate PS backbone modifications with small, well-tolerated2′-substitutions (2′-O-Methyl, 2′-Fluoro, 2′-Hydro) have been associatedwith highly stable siRNAs for applications in vivo, as reported bySoutschek et al. Nature 432:173-178 (2004); and 2′-O-Methylmodifications have been reported to be effective in improving stabilityas reported by Volkov, Oligonucleotides 19:191-202 (2009). With respectto decreasing the induction of innate immune responses, modifyingspecific sequences with 2′-O-Methyl, 2′-Fluoro, 2′-Hydro have beenreported to reduce TLR7/TLR8 interaction while generally preservingsilencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505(2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additionalmodifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine,5-methyluracil, and N6-methyladenosine have also been shown to minimizethe immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko,K. et al., Immunity 23:165-175 (2005).

As is also known in the art, and commercially available, a number ofconjugates can be applied to polynucleotides, such as RNAs, for useherein that can enhance their delivery and/or uptake by cells, includingfor example, cholesterol, tocopherol and folic acid, lipids, peptides,polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther.Deliv. 4:791-809 (2013), and references cited therein.

Codon-Optimization

In some embodiments, a polynucleotide encoding a site-directedpolypeptide is codon-optimized according to methods standard in the artfor expression in the cell containing the target DNA of interest. Forexample, if the intended target nucleic acid is in a human cell, a humancodon-optimized polynucleotide encoding Cas9 is contemplated for use forproducing the Cas9 polypeptide.

Complexes of a Genome-Targeting Nucleic Acid and a Site-DirectedPolypeptide

A genome-targeting nucleic acid interacts with a site-directedpolypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), therebyforming a complex. The genome-targeting nucleic acid guides thesite-directed polypeptide to a target nucleic acid.

RNPs

As stated previously, the site-directed polypeptide and genome-targetingnucleic acid may each be administered separately to a cell or a patient.On the other hand, the site-directed polypeptide may be pre-complexedwith one or more guide RNAs, or one or more crRNA together with atracrRNA. The pre-complexed material may then be administered to a cellor a patient. Such pre-complexed material is known as aribonucleoprotein particle (RNP).

Nucleic Acids Encoding System Components

In another aspect, the present disclosure provides a nucleic acidcomprising a nucleotide sequence encoding a genome-targeting nucleicacid of the disclosure, a site-directed polypeptide of the disclosure,and/or any nucleic acid or proteinaceous molecule necessary to carry outthe embodiments of the methods of the disclosure.

In some embodiments, the nucleic acid encoding a genome-targetingnucleic acid of the disclosure, a site-directed polypeptide of thedisclosure, and/or any nucleic acid or proteinaceous molecule necessaryto carry out the embodiments of the methods of the disclosure comprisesa vector (e.g., a recombinant expression vector).

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional nucleic acid segments can be ligated. Anothertype of vector is a viral vector, wherein additional nucleic acidsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome.

In some embodiments, vectors are capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “recombinant expression vectors”, or more simply“expression vectors”, which serve equivalent functions.

The term “operably linked” means that the nucleotide sequence ofinterest is linked to regulatory sequence(s) in a manner that allows forexpression of the nucleotide sequence. The term “regulatory sequence” isintended to include, for example, promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are well known in the art and are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells, and those that direct expressionof the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the target cell, the level ofexpression desired, and the like.

Expression vectors contemplated include, but are not limited to, viralvectors based on vaccinia virus, poliovirus, adenovirus,adeno-associated virus, SV40, herpes simplex virus, humanimmunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus,human immunodeficiency virus, myeloproliferative sarcoma virus, andmammary tumor virus) and other recombinant vectors. Other vectorscontemplated for eukaryotic target cells include, but are not limitedto, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).Additional vectors contemplated for eukaryotic target cells include, butare not limited to, the vectors pCTx-1, pCTx-2, and pCTx-3, which aredescribed in FIGS. 1A-1C. Other vectors may be used so long as they arecompatible with the host cell.

In some embodiments, a vector comprises one or more transcription and/ortranslation control elements. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationcontrol elements, including constitutive and inducible promoters,transcription enhancer elements, transcription terminators, etc. may beused in the expression vector. In some embodiments, the vector is aself-inactivating vector that either inactivates the viral sequences orthe components of the CRISPR machinery or other elements.

Non-limiting examples of suitable eukaryotic promoters (i.e., promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, humanelongation factor-1 promoter (EF1), a hybrid construct comprising thecytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter(CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1locus promoter (PGK), and mouse metallothionein-I.

For expressing small RNAs, including guide RNAs used in connection withCas endonuclease, various promoters such as RNA polymerase IIIpromoters, including for example U6 and H1, can be advantageous.Descriptions of and parameters for enhancing the use of such promotersare known in art, and additional information and approaches areregularly being described; see, e.g., Ma, H. et al., MolecularTherapy—Nucleic Acids 3, e161 (2014) doi:10.1038/mtna.2014.12.

The expression vector may also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector may also include appropriate sequences for amplifying expression.The expression vector may also include nucleotide sequences encodingnon-native tags (e.g., histidine tag, hemagglutinin tag, greenfluorescent protein, etc.) that are fused to the site-directedpolypeptide, thus resulting in a fusion protein.

In some embodiments, a promoter is an inducible promoter (e.g., a heatshock promoter, tetracycline-regulated promoter, steroid-regulatedpromoter, metal-regulated promoter, estrogen receptor-regulatedpromoter, etc.). In some embodiments, a promoter is a constitutivepromoter (e.g., CMV promoter, UBC promoter). In some embodiments, thepromoter is a spatially restricted and/or temporally restricted promoter(e.g., a tissue specific promoter, a cell type specific promoter, etc.).

In some embodiments, the nucleic acid encoding a genome-targetingnucleic acid of the disclosure and/or a site-directed polypeptide arepackaged into or on the surface of delivery vehicles for delivery tocells. Delivery vehicles contemplated include, but are not limited to,nanospheres, liposomes, quantum dots, nanoparticles, polyethylene glycolparticles, hydrogels, and micelles. As described in the art, a varietyof targeting moieties can be used to enhance the preferentialinteraction of such vehicles with desired cell types or locations.

Introduction of the complexes, polypeptides, and nucleic acids of thedisclosure into cells can occur by viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, nucleofection, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro-injection,nanoparticle-mediated nucleic acid delivery, and the like.

Delivery

Guide RNA polynucleotides (RNA or DNA) and/or endonucleasepolynucleotide(s) (RNA or DNA) can be delivered by viral or non-viraldelivery vehicles known in the art. Alternatively, endonucleasepolypeptide(s) may be delivered by non-viral delivery vehicles known inthe art, such as electroporation or lipid nanoparticles. In someembodiments, the DNA endonuclease may be delivered as one or morepolypeptides, either alone or pre-complexed with one or more guide RNAs,or one or more crRNA together with a tracrRNA.

Polynucleotides may be delivered by non-viral delivery vehiclesincluding, but not limited to, nanoparticles, liposomes,ribonucleoproteins, positively charged peptides, small moleculeRNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer andLieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses onnon-viral delivery vehicles for siRNA that are also useful for deliveryof other polynucleotides).

Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding anendonuclease, may be delivered to a cell or a patient by a lipidnanoparticle (LNP).

A LNP refers to any particle having a diameter of less than 1000 nm, 500nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.Alternatively, a nanoparticle may range in size from 1-1000 nm, 1-500nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

LNPs may be made from cationic, anionic, or neutral lipids. Neutrallipids, such as the fusogenic phospholipid DOPE or the membranecomponent cholesterol, may be included in LNPs as ‘helper lipids’ toenhance transfection activity and nanoparticle stability. Limitations ofcationic lipids include low efficacy owing to poor stability and rapidclearance, as well as the generation of inflammatory oranti-inflammatory responses.

LNPs may also be comprised of hydrophobic lipids, hydrophilic lipids, orboth hydrophobic and hydrophilic lipids.

Any lipid or combination of lipids that are known in the art may be usedto produce a LNP. Examples of lipids used to produce LNPs are: DOTMA,DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol,GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2),DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are:DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are:PEG-DMG, PEG-CerC14, and PEG-CerC20.

The lipids may be combined in any number of molar ratios to produce aLNP. In addition, the polynucleotide(s) may be combined with lipid(s) ina wide range of molar ratios to produce a LNP.

As stated previously, the site-directed polypeptide and genome-targetingnucleic acid may each be administered separately to a cell or a patient.On the other hand, the site-directed polypeptide may be pre-complexedwith one or more guide RNAs, or one or more crRNA together with atracrRNA. The pre-complexed material may then be administered to a cellor a patient. Such pre-complexed material is known as aribonucleoprotein particle (RNP).

RNA is capable of forming specific interactions with RNA or DNA. Whilethis property is exploited in many biological processes, it also comeswith the risk of promiscuous interactions in a nucleic acid-richcellular environment. One solution to this problem is the formation ofribonucleoprotein particles (RNPs), in which the RNA is pre-complexedwith an endonuclease. Another benefit of the RNP is protection of theRNA from degradation.

The endonuclease in the RNP may be modified or unmodified. Likewise, thegRNA, crRNA, tracrRNA, or sgRNA may be modified or unmodified. Numerousmodifications are known in the art and may be used.

The endonuclease and sgRNA are generally combined in a 1:1 molar ratio.Alternatively, the endonuclease, crRNA and tracrRNA are generallycombined in a 1:1:1 molar ratio. However, a wide range of molar ratiosmay be used to produce a RNP.

A recombinant adeno-associated virus (AAV) vector may be used fordelivery. Techniques to produce rAAV particles, in which an AAV genometo be packaged that includes the polynucleotide to be delivered, rep andcap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV requires that the followingcomponents are present within a single cell (denoted herein as apackaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes may be from any AAV serotype for which recombinant viruscan be derived, and may be from a different AAV serotype than the rAAVgenome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12,AAV-13 and AAV rh.74. Production of pseudotyped rAAV is disclosed in,for example, international patent application publication number WO01/83692. See Table 2.

TABLE 2 AAV Serotype Genbank Accession No. AAV-1 NC_002077.1 AAV-2NC_001401.2 AAV-3 NC_001729.1 AAV-3B AF028705.1 AAV-4 NC_001829.1 AAV-5NC_006152.1 AAV-6 AF028704.1 AAV-7 NC_006260.1 AAV-8 NC_006261.1 AAV-9AX753250.1 AAV-10 AY631965.1 AAV-11 AY631966.1 AAV-12 DQ813647.1 AAV-13EU285562.1

A method of generating a packaging cell involves creating a cell linethat stably expresses all of the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes, AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line is then infected with a helpervirus, such as adenovirus. The advantages of this method are that thecells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus orbaculovirus, rather than plasmids, to introduce rAAV genomes and/or repand cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos. 5,786,211;5,871,982; and 6,258,595.

AAV vector serotypes can be matched to target cell types. For example,the following exemplary cell types may be transduced by the indicatedAAV serotypes among others. See Table 3.

TABLE 3 Tissue/Cell Type Serotype Liver AAV3, AAV5, AAV8, AAV9 Skeletalmuscle AAV1, AAV7, AAV6, AAV8, AAV9 Central nervous system AAV5, AAV1,AAV4 RPE AAV5, AAV4 Photoreceptor cells AAV5 Lung AAV9 Heart AAV8Pancreas AAV8 Kidney AAV2, AAV8

In addition to adeno-associated viral vectors, other viral vectors canbe used. Such viral vectors include, but are not limited to, lentivirus,alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, EpsteinBarr virus, papovavirusr, poxvirus, vaccinia virus, and herpes simplexvirus.

In some embodiments, Cas9 mRNA, sgRNA targeting one or two loci inSERPINA1 genes, and donor DNA are each separately formulated into lipidnanoparticles, or are all co-formulated into one lipid nanoparticle, orco-formulated into two or more lipid nanoparticles.

In some embodiments, Cas9 mRNA is formulated in a lipid nanoparticle,while sgRNA and donor DNA are delivered in an AAV vector. In someembodiments, Cas9 mRNA and sgRNA are co-formulated in a lipidnanoparticle, while donor DNA is delivered in an AAV vector.

Options are available to deliver the Cas9 nuclease as a DNA plasmid, asmRNA or as a protein. The guide RNA can be expressed from the same DNA,or can also be delivered as an RNA. The RNA can be chemically modifiedto alter or improve its half-life, or decrease the likelihood or degreeof immune response. The endonuclease protein can be complexed with thegRNA prior to delivery. Viral vectors allow efficient delivery; splitversions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV,as can donors for HDR. A range of non-viral delivery methods also existthat can deliver each of these components, or non-viral and viralmethods can be employed in tandem. For example, nano-particles can beused to deliver the protein and guide RNA, while AAV can be used todeliver a donor DNA.

Exosomes

Exosomes, a type of microvesicle bound by phospholipid bilayer, can beused to deliver nucleic acids to specific tissue. Many different typesof cells within the body naturally secrete exosomes. Exosomes formwithin the cytoplasm when endosomes invaginate and formmultivesicular-endosomes (MVE). When the MVE fuses with the cellularmembrane, the exosomes are secreted in the extracellular space. Rangingbetween 30-120 nm in diameter, exosomes can shuttle various moleculesfrom one cell to another in a form of cell-to-cell communication. Cellsthat naturally produce exosomes, such as mast cells, can be geneticallyaltered to produce exosomes with surface proteins that target specifictissues, alternatively exosomes can be isolated from the bloodstream.Specific nucleic acids can be placed within the engineered exosomes withelectroporation. When introduced systemically, the exosomes can deliverthe nucleic acids to the specific target tissue.

Genetically Modified Cells

The term “genetically modified cell” refers to a cell that comprises atleast one genetic modification introduced by genome editing (e.g., usingthe CRISPR/Cas system). In some ex vivo embodiments herein, thegenetically modified cell is a genetically modified progenitor cell. Insome in vivo embodiments herein, the genetically modified cell is agenetically modified liver cell. A genetically modified cell comprisingan exogenous genome-targeting nucleic acid and/or an exogenous nucleicacid encoding a genome-targeting nucleic acid is contemplated herein.

The term “control treated population” describes a population of cellsthat has been treated with identical media, viral induction, nucleicacid sequences, temperature, confluency, flask size, pH, etc., with theexception of the addition of the genome editing components. Any methodknown in the art can be used to measure restoration of SERPINA1 gene orprotein expression or activity, for example, Western Blot analysis ofthe AAT protein or quantifying SERPINA1 mRNA.

The term “isolated cell” refers to a cell that has been removed from anorganism in which it was originally found, or a descendant of such acell. Optionally, the cell has been cultured in vitro, e.g., underdefined conditions or in the presence of other cells. Optionally, thecell is later introduced into a second organism or re-introduced intothe organism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells refers to a population of cells that has been removed andseparated from a mixed or heterogeneous population of cells. In someembodiments, an isolated population is a substantially pure populationof cells, as compared to the heterogeneous population from which thecells were isolated or enriched. In some embodiments, the isolatedpopulation is an isolated population of human progenitor cells, e.g., asubstantially pure population of human progenitor cells, as compared toa heterogeneous population of cells comprising human progenitor cellsand cells from which the human progenitor cells were derived.

The term “substantially enhanced,” with respect to a particular cellpopulation, refers to a population of cells in which the occurrence of aparticular type of cell is increased relative to pre-existing orreference levels, by at least 2-fold, at least 3-, at least 4-, at least5-, at least 6-, at least 7-, at least 8-, at least 9, at least 10-, atleast 20-, at least 50-, at least 100-, at least 400-, at least 1000-,at least 5000-, at least 20000-, at least 100000- or more folddepending, e.g., on the desired levels of such cells for amelioratingAATD.

The term “substantially enriched” with respect to a particular cellpopulation, refers to a population of cells that is at least about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or morewith respect to the cells making up a total cell population.

The terms “substantially enriched” or “substantially pure” with respectto a particular cell population, refers to a population of cells that isat least about 75%, at least about 85%, at least about 90%, or at leastabout 95% pure, with respect to the cells making up a total cellpopulation. That is, the terms “substantially pure” or “essentiallypurified,” with regard to a population of progenitor cells, refers to apopulation of cells that contain fewer than about 20%, about 15%, about10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about3%, about 2%, about 1%, or less than 1%, of cells that are notprogenitor cells as defined by the terms herein.

Differentiation of Genome Edited IPSCs into Hepatocytes

Another step of the ex vivo methods of the invention involvesdifferentiating the genome edited iPSCs into hepatocytes. Thedifferentiating step may be performed according to any method known inthe art. For example, hiPSC are differentiated into definitive endodermusing various treatments, including activin and B27 supplement (LifeTechnology). The definitive endoderm is further differentiated intohepatocyte, the treatment includes: FGF4, HGF, BMP2, BMP4, Oncostatin M,Dexametason, etc. (Duan et al, STEM CELLS; 2010; 28:674-686, Ma et al,STEM CELLS TRANSLATIONAL MEDICINE 2013; 2:409-419).

Differentiation of Genome Edited Mesenchymal Stem Cells into Hepatocytes

Another step of the ex vivo methods of the invention involvesdifferentiating the genome edited mesenchymal stem cells intohepatocytes. The differentiating step may be performed according to anymethod known in the art. For example, hMSC are treated with variousfactors and hormones, including insulin, transferrin, FGF4, HGF, bileacids (Sawitza I et al, Sci Rep. 2015; 5: 13320).

Implanting Cells into Patients

Another step of the ex vivo methods of the invention involves implantingthe hepatocytes into patients. This implanting step may be accomplishedusing any method of implantation known in the art. For example, thegenetically modified cells may be injected directly in the patient'sliver or otherwise administered to the patient.

Another step of the ex vivo methods of the invention involves implantingthe progenitor cells or primary hepatocytes into patients. Thisimplanting step may be accomplished using any method of implantationknown in the art. For example, the genetically modified cells may beinjected directly in the patients liver or otherwise administered to thepatient. The genetically modified cells may be purified ex vivo using aselected marker.

Pharmaceutically Acceptable Carriers

The ex vivo methods of administering progenitor cells to a subjectcontemplated herein involve the use of therapeutic compositionscomprising progenitor cells.

Therapeutic compositions contain a physiologically tolerable carriertogether with the cell composition, and optionally at least oneadditional bioactive agent as described herein, dissolved or dispersedtherein as an active ingredient. In some embodiments, the therapeuticcomposition is not substantially immunogenic when administered to amammal or human patient for therapeutic purposes, unless so desired.

In general, the progenitor cells described herein are administered as asuspension with a pharmaceutically acceptable carrier. One of skill inthe art will recognize that a pharmaceutically acceptable carrier to beused in a cell composition will not include buffers, compounds,cryopreservation agents, preservatives, or other agents in amounts thatsubstantially interfere with the viability of the cells to be deliveredto the subject. A formulation comprising cells can include e.g., osmoticbuffers that permit cell membrane integrity to be maintained, andoptionally, nutrients to maintain cell viability or enhance engraftmentupon administration. Such formulations and suspensions are known tothose of skill in the art and/or can be adapted for use with theprogenitor cells, as described herein, using routine experimentation.

A cell composition can also be emulsified or presented as a liposomecomposition, provided that the emulsification procedure does notadversely affect cell viability. The cells and any other activeingredient can be mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient, and in amountssuitable for use in the therapeutic methods described herein.

Additional agents included in a cell composition can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids, such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases, such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in the cellcompositions that is effective in the treatment of a particular disorderor condition will depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques.

Administration & Efficacy

The terms “administering,” “introducing” and “transplanting” are usedinterchangeably in the context of the placement of cells, e.g.,progenitor cells, into a subject, by a method or route that results inat least partial localization of the introduced cells at a desired site,such as a site of injury or repair, such that a desired effect(s) isproduced. The cells e.g., progenitor cells, or their differentiatedprogeny can be administered by any appropriate route that results indelivery to a desired location in the subject where at least a portionof the implanted cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years, or even the life time of the patient, i.e.,long-term engraftment. For example, in some embodiments describedherein, an effective amount of myogenic progenitor cells is administeredvia a systemic route of administration, such as an intraperitoneal orintravenous route.

The terms “individual”, “subject,” “host” and “patient” are usedinterchangeably herein and refer to any subject for whom diagnosis,treatment or therapy is desired. In some embodiments, the subject is amammal. In some embodiments, the subject is a human being.

When provided prophylactically, progenitor cells described herein can beadministered to a subject in advance of any symptom of AATD, e.g., priorto the development of emphysema, airflow obstruction, chronicbronchitis, or liver disease. Accordingly, the prophylacticadministration of a liver progenitor cell population serves to preventAATD.

When provided therapeutically, liver progenitor cells are provided at(or after) the onset of a symptom or indication of AATD, e.g., upon theonset of lung or liver disease.

In some embodiments described herein, the liver progenitor cellpopulation being administered according to the methods described hereincomprises allogeneic liver progenitor cells obtained from one or moredonors. “Allogeneic” refers to a liver progenitor cell or biologicalsamples comprising liver progenitor cells or biological samples obtainedfrom one or more different donors of the same species, where the genesat one or more loci are not identical. For example, a liver progenitorcell population being administered to a subject can be derived from onemore unrelated donor subjects, or from one or more non-identicalsiblings. In some embodiments, syngeneic liver progenitor cellpopulations can be used, such as those obtained from geneticallyidentical animals, or from identical twins. In other embodiments, theliver progenitor cells are autologous cells; that is, the liverprogenitor cells are obtained or isolated from a subject andadministered to the same subject, i.e., the donor and recipient are thesame.

In one embodiment, the term “effective amount” refers to the amount of apopulation of progenitor cells or their progeny needed to prevent oralleviate at least one or more signs or symptoms of AATD, and relates toa sufficient amount of a composition to provide the desired effect,e.g., to treat a subject having AATD. The term “therapeuticallyeffective amount” therefore refers to an amount of progenitor cells or acomposition comprising progenitor cells that is sufficient to promote aparticular effect when administered to a typical subject, such as onewho has or is at risk for AATD. An effective amount would also includean amount sufficient to prevent or delay the development of a symptom ofthe disease, alter the course of a symptom of the disease (for examplebut not limited to, slow the progression of a symptom of the disease),or reverse a symptom of the disease. It is understood that for any givencase, an appropriate “effective amount” can be determined by one ofordinary skill in the art using routine experimentation.

For use in the various embodiments described herein, an effective amountof progenitor cells comprises at least 10² progenitor cells, at least5×10² progenitor cells, at least 10³ progenitor cells, at least 5×10³progenitor cells, at least 10⁴ progenitor cells, at least 5×10⁴progenitor cells, at least 10⁵ progenitor cells, at least 2×10⁵progenitor cells, at least 3×10⁵ progenitor cells, at least 4×10⁵progenitor cells, at least 5×10⁵ progenitor cells, at least 6×10⁵progenitor cells, at least 7×10⁵ progenitor cells, at least 8×10⁵progenitor cells, at least 9×10⁵ progenitor cells, at least 1×10⁶progenitor cells, at least 2×10⁶ progenitor cells, at least 3×10⁶progenitor cells, at least 4×10⁶ progenitor cells, at least 5×10⁶progenitor cells, at least 6×10⁶ progenitor cells, at least 7×10⁶progenitor cells, at least 8×10⁶ progenitor cells, at least 9×10⁶progenitor cells, or multiples thereof. The progenitor cells are derivedfrom one or more donors, or are obtained from an autologous source. Insome embodiments described herein, the progenitor cells are expanded inculture prior to administration to a subject in need thereof.

Modest and incremental increases in the levels of functional AATexpressed in cells of patients having AATD can be beneficial forameliorating one or more symptoms of the disease, for increasinglong-term survival, and/or for reducing side effects associated withother treatments. Upon administration of such cells to human patients,the presence of liver progenitors that are producing increased levels offunctional AAT is beneficial. In some embodiments, effective treatmentof a subject gives rise to at least about 3%, 5% or 7% functional AATrelative to total AAT in the treated subject. In some embodiments,functional AAT will be at least about 10% of total AAT. In someembodiments, functional AAT will be at least about 20% to 30% of totalAAT. Similarly, the introduction of even relatively limitedsubpopulations of cells having significantly elevated levels offunctional AAT can be beneficial in various patients because in somesituations normalized cells will have a selective advantage relative todiseased cells. However, even modest levels of liver progenitors withelevated levels of functional AAT can be beneficial for ameliorating oneor more aspects of AATD in patients. In some embodiments, about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about80%, about 90% or more of the liver progenitors in patients to whom suchcells are administered are producing increased levels of functional AAT.

“Administered” refers to the delivery of a progenitor cell compositioninto a subject by a method or route that results in at least partiallocalization of the cell composition at a desired site. A cellcomposition can be administered by any appropriate route that results ineffective treatment in the subject, i.e. administration results indelivery to a desired location in the subject where at least a portionof the composition delivered, i.e. at least 1×10⁴ cells are delivered tothe desired site for a period of time. Modes of administration includeinjection, infusion, instillation, or ingestion. “Injection” includes,without limitation, intravenous, intramuscular, intra-arterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. In someembodiments, the route is intravenous. For the delivery of cells,administration by injection or infusion can be made.

In one embodiment, the cells are administered systemically. The phrases“systemic administration,” “administered systemically”, “peripheraladministration” and “administered peripherally” refer to theadministration of a population of progenitor cells other than directlyinto a target site, tissue, or organ, such that it enters, instead, thesubject's circulatory system and, thus, is subject to metabolism andother like processes.

The efficacy of a treatment comprising a composition for the treatmentof AATD can be determined by the skilled clinician. However, a treatmentis considered “effective treatment,” if any one or all of the signs orsymptoms of, as but one example, levels of functional AAT are altered ina beneficial manner (e.g., increased by at least 10%), or otherclinically accepted symptoms or markers of disease are improved orameliorated. Efficacy can also be measured by failure of an individualto worsen as assessed by hospitalization or need for medicalinterventions (e.g., chronic obstructive pulmonary disease, orprogression of the disease is halted or at least slowed). Methods ofmeasuring these indicators are known to those of skill in the art and/ordescribed herein. Treatment includes any treatment of a disease in anindividual or an animal (some non-limiting examples include a human, ora mammal) and includes: (1) inhibiting the disease, e.g., arresting, orslowing the progression of symptoms; or (2) relieving the disease, e.g.,causing regression of symptoms; and (3) preventing or reducing thelikelihood of the development of symptoms.

The treatment according to the present invention ameliorates one or moresymptoms associated with AATD by increasing the amount of functional AATin the individual. Early signs typically associated with AATD, includefor example, emphysema, airflow obstruction, chronic bronchitis, orliver disease (obstructive jaundice and increased aminotransferaselevels in early life, and fibrosis and cirrhosis in the absence of ahistory of liver complications during childhood).

Kits

The present disclosure provides kits for carrying out the methods of theinvention. A kit can include one or more of a genome-targeting nucleicacid, a polynucleotide encoding a genome-targeting nucleic acid, asite-directed polypeptide, a polynucleotide encoding a site-directedpolypeptide, and/or any nucleic acid or proteinaceous molecule necessaryto carry out the embodiments of the methods of the invention, or anycombination thereof.

In some embodiments, a kit comprises: (1) a vector comprising anucleotide sequence encoding a genome-targeting nucleic acid, and (2)the site-directed polypeptide or a vector comprising a nucleotidesequence encoding the site-directed polypeptide and (3) a reagent forreconstitution and/or dilution of the vector(s) and or polypeptide.

In some embodiments, a kit comprises: (1) a vector comprising (i) anucleotide sequence encoding a genome-targeting nucleic acid, and (ii) anucleotide sequence encoding the site-directed polypeptide and (2) areagent for reconstitution and/or dilution of the vector.

In some embodiments of any of the above kits, the kit comprises asingle-molecule guide genome-targeting nucleic acid. In some embodimentsof any of the above kits, the kit comprises a double-moleculegenome-targeting nucleic acid. In some embodiments of any of the abovekits, the kit comprises two or more double-molecule guides orsingle-molecule guides. In some embodiments, the kits comprise a vectorthat encodes the nucleic acid targeting nucleic acid.

In some embodiments of any of the above kits, the kit can furthercomprise a polynucleotide to be inserted to effect the desired geneticmodification.

Components of a kit may be in separate containers, or combined in asingle container.

In some embodiments, a kit described above further comprises one or moreadditional reagents, where such additional reagents are selected from abuffer, a buffer for introducing a polypeptide or polynucleotide into acell, a wash buffer, a control reagent, a control vector, a control RNApolynucleotide, a reagent for in vitro production of the polypeptidefrom DNA, adaptors for sequencing and the like. A buffer can be astabilization buffer, a reconstituting buffer, a diluting buffer, or thelike. In some embodiments, a kit can also include one or more componentsthat may be used to facilitate or enhance the on-target binding or thecleavage of DNA by the endonuclease, or improve the specificity oftargeting.

In addition to the above-mentioned components, a kit can further includeinstructions for using the components of the kit to practice themethods. The instructions for practicing the methods are generallyrecorded on a suitable recording medium. For example, the instructionsmay be printed on a substrate, such as paper or plastic, etc. Theinstructions may be present in the kits as a package insert, in thelabeling of the container of the kit or components thereof (i.e.,associated with the packaging or subpackaging), etc. The instructionscan be present as an electronic storage data file present on a suitablecomputer readable storage medium, e.g. CD-ROM, diskette, flash drive,etc. In some instances, the actual instructions are not present in thekit, but means for obtaining the instructions from a remote source (e.g.via the Internet), can be provided. An example of this embodiment is akit that includes a web address where the instructions can be viewedand/or from which the instructions can be downloaded. As with theinstructions, this means for obtaining the instructions can be recordedon a suitable substrate.

Guide RNA Formulation

Guide RNAs of the invention are formulated with pharmaceuticallyacceptable excipients such as carriers, solvents, stabilizers,adjuvants, diluents, etc., depending upon the particular mode ofadministration and dosage form. Guide RNA compositions are generallyformulated to achieve a physiologically compatible pH, and range from apH of about 3 to a pH of about 11, about pH 3 to about pH 7, dependingon the formulation and route of administration. In some embodiments, thepH is adjusted to a range from about pH 5.0 to about pH 8. In someembodiments, the compositions comprise a therapeutically effectiveamount of at least one compound as described herein, together with oneor more pharmaceutically acceptable excipients. Optionally, thecompositions comprise a combination of the compounds described herein,or may include a second active ingredient useful in the treatment orprevention of bacterial growth (for example and without limitation,anti-bacterial or anti-microbial agents), or may include a combinationof reagents of the invention.

Suitable excipients include, for example, carrier molecules that includelarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and inactive virus particles. Otherexemplary excipients include antioxidants (for example and withoutlimitation, ascorbic acid), chelating agents (for example and withoutlimitation, EDTA), carbohydrates (for example and without limitation,dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose),stearic acid, liquids (for example and without limitation, oils, water,saline, glycerol and ethanol), wetting or emulsifying agents, pHbuffering substances, and the like.

Other Possible Therapeutic Approaches

Gene editing can be conducted using nucleases engineered to targetspecific sequences. To date there are four major types of nucleases:meganucleases and their derivatives, zinc finger nucleases (ZFNs),transcription activator like effector nucleases (TALENs), andCRISPR-Cas9 nuclease systems. The nuclease platforms vary in difficultyof design, targeting density and mode of action, particularly as thespecificity of ZFNs and TALENs is through protein-DNA interactions,while RNA-DNA interactions primarily guide Cas9. Cas9 cleavage alsorequires an adjacent motif, the PAM, which differs between differentCRISPR systems. Cas9 from Streptococcus pyogenes cleaves using a NRGPAM, CRISPR from Neisseria meningitidis can cleave at sites with PAMsincluding NNNNGATT, NNNNNGTTT and NNNNGCTT. A number of other Cas9orthologs target protospacer adjacent to alternative PAMs.

CRISPR endonucleases, such as Cas9, can be used in the methods of theinvention. However, the teachings described herein, such as therapeutictarget sites, could be applied to other forms of endonucleases, such asZFNs, TALENs, HEs, or MegaTALs, or using combinations of nucleases.However, in order to apply the teachings of the present invention tosuch endonucleases, one would need to, among other things, engineerproteins directed to the specific target sites.

Additional binding domains can be fused to the Cas9 protein to increasespecificity. The target sites of these constructs would map to theidentified gRNA specified site, but would require additional bindingmotifs, such as for a zinc finger domain. In the case of Mega-TAL, ameganuclease can be fused to a TALE DNA-binding domain. The meganucleasedomain can increase specificity and provide the cleavage. Similarly,inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain andrequire the sgRNA/Cas9 target site and adjacent binding site for thefused DNA-binding domain. This likely would require some proteinengineering of the dCas9, in addition to the catalytic inactivation, todecrease binding without the additional binding site.

Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are modular proteins comprised of anengineered zinc finger DNA binding domain linked to the catalytic domainof the type II endonuclease Fokl. Because Fokl functions only as adimer, a pair of ZFNs must be engineered to bind to cognate target“half-site” sequences on opposite DNA strands and with precise spacingbetween them to enable the catalytically active Fokl dimer to form. Upondimerization of the Fokl domain, which itself has no sequencespecificity per se, a DNA double-strand break is generated between theZFN half-sites as the initiating step in genome editing.

The DNA binding domain of each ZFN is typically comprised of 3-6 zincfingers of the abundant Cys2-His2 architecture, with each fingerprimarily recognizing a triplet of nucleotides on one strand of thetarget DNA sequence, although cross-strand interaction with a fourthnucleotide also can be important. Alteration of the amino acids of afinger in positions that make key contacts with the DNA alters thesequence specificity of a given finger. Thus, a four-finger zinc fingerprotein will selectively recognize a 12 bp target sequence, where thetarget sequence is a composite of the triplet preferences contributed byeach finger, although triplet preference can be influenced to varyingdegrees by neighboring fingers. An important aspect of ZFNs is that theycan be readily re-targeted to almost any genomic address simply bymodifying individual fingers, although considerable expertise isrequired to do this well. In most applications of ZFNs, proteins of 4-6fingers are used, recognizing 12-18 bp respectively. Hence, a pair ofZFNs will typically recognize a combined target sequence of 24-36 bp,not including the 5-7 bp spacer between half-sites. The binding sitescan be separated further with larger spacers, including 15-17 bp. Atarget sequence of this length is likely to be unique in the humangenome, assuming repetitive sequences or gene homologs are excludedduring the design process. Nevertheless, the ZFN protein-DNAinteractions are not absolute in their specificity so off-target bindingand cleavage events do occur, either as a heterodimer between the twoZFNs, or as a homodimer of one or the other of the ZFNs. The latterpossibility has been effectively eliminated by engineering thedimerization interface of the Fokl domain to create “plus” and “minus”variants, also known as obligate heterodimer variants, which can onlydimerize with each other, and not with themselves. Forcing the obligateheterodimer prevents formation of the homodimer. This has greatlyenhanced specificity of ZFNs, as well as any other nuclease that adoptsthese Fokl variants.

A variety of ZFN-based systems have been described in the art,modifications thereof are regularly reported, and numerous referencesdescribe rules and parameters that are used to guide the design of ZFNs;see, e.g., Segal et al., Proc Natl Acad Sci USA 96(6):2758-63 (1999);Dreier B et al., J Mol Biol. 303(4):489-502 (2000); Liu Q et al., J BiolChem. 277(6):3850-6 (2002); Dreier et al., J Biol Chem 280(42):35588-97(2005); and Dreier et al., J Biol Chem. 276(31):29466-78 (2001).

Transcription Activator-Like Effector Nucleases (TALENs)

TALENs represent another format of modular nucleases whereby, as withZFNs, an engineered DNA binding domain is linked to the Fokl nucleasedomain, and a pair of TALENs operate in tandem to achieve targeted DNAcleavage. The major difference from ZFNs is the nature of the DNAbinding domain and the associated target DNA sequence recognitionproperties. The TALEN DNA binding domain derives from TALE proteins,which were originally described in the plant bacterial pathogenXanthomonas sp. TALEs are comprised of tandem arrays of 33-35 amino acidrepeats, with each repeat recognizing a single basepair in the targetDNA sequence that is typically up to 20 bp in length, giving a totaltarget sequence length of up to 40 bp. Nucleotide specificity of eachrepeat is determined by the repeat variable diresidue (RVD), whichincludes just two amino acids at positions 12 and 13. The bases guanine,adenine, cytosine and thymine are predominantly recognized by the fourRVDs: Asn-Asn, Asn-Ille, His-Asp and Asn-Gly, respectively. Thisconstitutes a much simpler recognition code than for zinc fingers, andthus represents an advantage over the latter for nuclease design.Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs arenot absolute in their specificity, and TALENs have also benefited fromthe use of obligate heterodimer variants of the Fokl domain to reduceoff-target activity.

Additional variants of the Fokl domain have been created that aredeactivated in their catalytic function. If one half of either a TALENor a ZFN pair contains an inactive Fokl domain, then only single-strandDNA cleavage (nicking) will occur at the target site, rather than a DSB.The outcome is comparable to the use of CRISPR/Cas9 “nickase” mutants inwhich one of the Cas9 cleavage domains has been deactivated. DNA nickscan be used to drive genome editing by HDR, but at lower efficiency thanwith a DSB. The main benefit is that off-target nicks are quickly andaccurately repaired, unlike the DSB, which is prone to NHEJ-mediatedmis-repair.

A variety of TALEN-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., Boch, Science326(5959):1509-12 (2009); Mak et al., Science 335(6069):716-9 (2012);and Moscou et al., Science 326(5959):1501 (2009). The use of TALENsbased on the “Golden Gate” platform, or cloning scheme, has beendescribed by multiple groups; see, e.g., Cermak et al., Nucleic AcidsRes. 39(12):e82 (2011); Li et al., Nucleic Acids Res. 39(14):6315-25(2011); Weber et al., PLoS One. 6(2):e16765 (2011); Wang et al., J GenetGenomics 41(6):339-47, Epub 2014 May 17 (2014); and Cermak T et al.,Methods Mol Biol. 1239:133-59 (2015).

Homing Endonucleases

Homing endonucleases (HEs) are sequence-specific endonucleases that havelong recognition sequences (14-44 base pairs) and cleave DNA with highspecificity—often at sites unique in the genome. There are at least sixknown families of HEs as classified by their structure, includingLAGLIDADG (SEQ ID NO: 68,299), GIY-YIG, His-Cis box, H-N-H, PD-(D/E)xK,and Vsr-like that are derived from a broad range of hosts, includingeukarya, protists, bacteria, archaea, cyanobacteria and phage. As withZFNs and TALENs, HEs can be used to create a DSB at a target locus asthe initial step in genome editing. In addition, some natural andengineered HEs cut only a single strand of DNA, thereby functioning assite-specific nickases. The large target sequence of HEs and thespecificity that they offer have made them attractive candidates tocreate site-specific DSBs.

A variety of HE-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., the reviews bySteentoft et al., Glycobiology 24(8):663-80 (2014); Belfort andBonocora, Methods Mol Biol. 1123:1-26 (2014); Hafez and Hausner, Genome55(8):553-69 (2012); and references cited therein.

MegaTAL/Tev-mTALEN/MegaTev

As further examples of hybrid nucleases, the MegaTAL platform andTev-mTALEN platform use a fusion of TALE DNA binding domains andcatalytically active HEs, taking advantage of both the tunable DNAbinding and specificity of the TALE, as well as the cleavage sequencespecificity of the HE; see, e.g., Boissel et al., NAR 42: 2591-2601(2014); Kleinstiver et al., G3 4:1155-65 (2014); and Boissel andScharenberg, Methods Mol. Biol. 1239: 171-96 (2015).

In a further variation, the MegaTev architecture is the fusion of ameganuclease (Mega) with the nuclease domain derived from the GIY-YIGhoming endonuclease I-Tevl (Tev). The two active sites are positioned˜30 bp apart on a DNA substrate and generate two DSBs withnon-compatible cohesive ends; see, e.g., Wolfs et al., NAR 42, 8816-29(2014). It is anticipated that other combinations of existingnuclease-based approaches will evolve and be useful in achieving thetargeted genome modifications described herein.

dCas9-Fokl and Other Nucleases

Combining the structural and functional properties of the nucleaseplatforms described above offers a further approach to genome editingthat can potentially overcome some of the inherent deficiencies. As anexample, the CRISPR genome editing system typically uses a single Cas9endonuclease to create a DSB. The specificity of targeting is driven bya 20 or 22 nucleotide sequence in the guide RNA that undergoesWatson-Crick base-pairing with the target DNA (plus an additional 2bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 fromS. pyogenes). Such a sequence is long enough to be unique in the humangenome, however, the specificity of the RNA/DNA interaction is notabsolute, with significant promiscuity sometimes tolerated, particularlyin the 5′ half of the target sequence, effectively reducing the numberof bases that drive specificity. One solution to this has been tocompletely deactivate the Cas9 catalytic function—retaining only theRNA-guided DNA binding function—and instead fusing a Fokl domain to thedeactivated Cas9; see, e.g., Tsai et al., Nature Biotech 32: 569-76(2014); and Guilinger et al., Nature Biotech. 32: 577-82 (2014). BecauseFokl must dimerize to become catalytically active, two guide RNAs arerequired to tether two Cas9-Fokl fusions in close proximity to form thedimer and cleave DNA. This essentially doubles the number of bases inthe combined target sites, thereby increasing the stringency oftargeting by CRISPR-based systems.

As further example, fusion of the TALE DNA binding domain to acatalytically active HE, such as I-Tevl, takes advantage of both thetunable DNA binding and specificity of the TALE, as well as the cleavagesequence specificity of I-Tevl, with the expectation that off-targetcleavage may be further reduced.

Methods and Compositions of the Invention

Accordingly, the present disclosure relates in particular to thefollowing non-limiting inventions: In a first method, Method 1, thepresent disclosure provides a method for editing the SERPINA1 gene in ahuman cell by genome editing, the method comprising the step ofintroducing into the human cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the SERPINA1 gene or otherDNA sequences that encode regulatory elements of the SERPINA1 gene thatresults in a permanent deletion, insertion, or correction or modulationof expression or function of one or more mutations or exons within ornear or affecting the expression or function of the SERPINA1 gene orother DNA sequences that encode regulatory elements of the SERPINA1 geneand restoration of alpha-1-antitrypsin (AAT) protein activity.

In another method, Method 2, the present disclosure provides a methodfor inserting a SERPINA1 gene in a human cell by genome editing, themethod comprising introducing into the human cell one or moredeoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear a safe harbor locus that results in a permanent insertion of theSERPINA1 gene or minigene, and results in restoration of AAT activity.

In another method, Method 3, the present disclosure provides an ex vivomethod for treating a patient with alpha-1 antitrypsin deficiency (AATD)comprising the steps of: i) creating a patient specific inducedpluripotent stem cell (iPSC); ii) editing within or near a SERPINA1 geneor other DNA sequences that encode regulatory elements of the SERPINA1gene of the iPSC, or within or near a safe harbor locus of the iPSC;iii) differentiating the genome edited iPSC into a hepatocyte; and iv)implanting the hepatocyte into the patient.

In another method, Method 4, the present disclosure provides the methodof Method 3, wherein the creating step comprises: a) isolating a somaticcell from the patient; and b) introducing a set ofpluripotency-associated genes into the somatic cell to induce thesomatic cell to become a pluripotent stem cell.

In another method, Method 5, the present disclosure provides the methodof Method 4, wherein the somatic cell is a fibroblast.

In another method, Method 6, the present disclosure provides the methodof Method 4, wherein the set of pluripotency-associated genes is one ormore of the genes selected from the group consisting of OCT4, SOX2,KLF4, Lin28, NANOG and cMYC.

In another method, Method 7, the present disclosure provides the methodof any one of Methods 3-6, wherein the editing step comprisesintroducing into the iPSC one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the SERPINA1 gene or otherDNA sequences that encode regulatory elements of the SERPINA1 gene thatresults in permanent deletion, insertion, or correction or modulation ofexpression or function of one or more mutations or exons within or nearor affecting the expression or function of the SERPINA1 gene or otherDNA sequences that encode regulatory elements of the SERPINA1 gene, orwithin or near a safe harbor locus that results in permanent insertionof the SERPINA1 or minigene, and restoration of alpha-1-antitrypsin(AAT) protein activity.

In another method, Method 8, the present disclosure provides the methodof Method 7, wherein the safe harbor locus is selected from the groupconsisting of AAVS1 (PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX(F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, and TTR.

In another method, Method 9, the present disclosure provides the methodof any one of Methods 3-8, wherein the differentiating step comprisesone or more of the following to differentiate the genome edited iPSCinto a hepatocyte: contacting the genome edited iPSC with one or more ofactivin, B27 supplement, FGF4, HGF, BMP2, BMP4, Oncostatin M,Dexametason.

In another method, Method 10, the present disclosure provides the methodof any one of Methods 3-9, wherein the implanting step comprisesimplanting the hepatocyte into the patient by transplantation, localinjection, or systemic infusion, or combinations thereof.

In another method, Method 11, the present disclosure provides an ex vivomethod for treating a patient with alpha-1 antitrypsin deficiency(AATD), the method comprising the steps of: i) performing a biopsy ofthe patient's liver, ii) isolating a liver specific progenitor cell orprimary hepatocyte; iii) editing within or near the SERPINA1 gene orother DNA sequences that encode regulatory elements of the SERPINA1 geneof the progenitor cell or primary hepatocyte or editing within or near asafe harbor locus of the progenitor cell or primary hepatocyte; and iv)implanting the genome-edited progenitor cell or primary hepatocyte intothe patient.

In another method, Method 12, the present disclosure provides the methodof Method 11, wherein the isolating step comprises: perfusion of freshliver tissues with digestion enzymes, cell differential centrifugation,cell culturing, and combinations thereof.

In another method, Method 13, the present disclosure provides the methodof any one of Methods 11-12, wherein the editing step comprisesintroducing into the progenitor cell or primary hepatocyte one or moredeoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the SERPINA1 gene or other DNA sequences that encode regulatoryelements of the SERPINA1 gene that results in a permanent deletion,insertion, correction, or modulation of expression or function of one ormore mutations or exons within or near or affecting the expression orfunction the SERPINA1 gene or other DNA sequences that encode regulatoryelements of the SERPINA1 gene, or within or near a safe harbor locusthat results in permanent insertion of the SERPINA1 gene or minigene,and restoration of alpha-1-antitrypsin (AAT) protein activity.

In another method, Method 14, the present disclosure provides the methodof Method 13, wherein the safe harbor locus is selected from the groupconsisting of AAVS1 (PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX(F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, and TTR.

In another method, Method 15, the present disclosure provides the methodof any one of Methods 11-14, wherein the implanting step comprisesimplanting the genome-edited progenitor cell or primary hepatocyte intothe patient by transplantation, local injection, or systemic infusion,or combinations thereof.

In another method, Method 16, the present disclosure provides an ex vivomethod for treating a patient with alpha-1 antitrypsin deficiency(AATD), the method comprising the steps of: i) performing a biopsy ofthe patients bone marrow; ii) isolating a mesenchymal stem cell from thepatient; iii) editing within or near the SERPINA1 gene or other DNAsequences that encode regulatory elements of the SERPINA1 gene of themesenchymal stem cell or editing within or near a safe harbor locus ofthe mesenchymal stem cell; iv) differentiating the genome-editedmesenchymal stem cell into a hepatocyte; and v) implanting thehepatocyte into the patient.

In another method, Method 17, the present disclosure provides the methodof Method 16, wherein the mesenchymal stem cell is isolated from thepatient's bone marrow or peripheral blood.

In another method, Method 18, the present disclosure provides the methodof Method 16, wherein the isolating step comprises: aspiration of bonemarrow and isolation of mesenchymal cells by density centrifugationusing Percoll™.

In another method, Method 19, the present disclosure provides the methodof any one of Methods 16-18, wherein the editing step comprisesintroducing into the mesenchymal stem cell one or more deoxyribonucleicacid (DNA) endonucleases to effect one or more single-strand breaks(SSBs) or double-strand breaks (DSBs) within or near the SERPINA1 geneor other DNA sequences that encode regulatory elements of the SERPINA1gene that results in permanent deletion, insertion, correction, ormodulation of expression or function of one or more mutations or exonswithin or near or affecting the expression or function of the SERPINA1gene or other DNA sequences that encode regulatory elements of theSERPINA1 gene, or within or near a safe harbor locus that results inpermanent insertion of the SERPINA1 gene or minigene, and restoration ofalpha-1-antitrypsin (AAT) protein activity.

In another method, Method 20, the present disclosure provides the methodof Method 19, wherein the safe harbor locus is selected from the groupconsisting of AAVS1 (PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX(F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1, TF, and TTR.

In another method, Method 21, the present disclosure provides the methodof any one of Methods 16-19, wherein the differentiating step comprisesone or more of the following to differentiate the genome edited stemcell into a hepatocyte: contacting the genome edited mesenchymal stemcell with one or more of insulin, transferrin, FGF4, HGF, or bile acids.

In another method, Method 22, the present disclosure provides the methodof any one of Methods 16-21, wherein the implanting step comprisesimplanting the hepatocyte into the patient by transplantation, localinjection, or systemic infusion, or combinations thereof.

In another method, Method 23, the present disclosure provides an in vivomethod for treating a patient with alpha-1 antitrypsin deficiency(AATD), the method comprising the step of editing the SERPINA1 gene in acell of the patient, or other DNA sequences that encode regulatoryelements of the SERPINA1 gene, or editing within or near a safe harborlocus in a cell of the patient.

In another method, Method 24, the present disclosure provides the methodof Method 23, wherein the editing step comprises introducing into thecell one or more deoxyribonucleic acid (DNA) endonucleases to effect oneor more single-strand breaks (SSBs) or double-strand breaks (DSBs)within or near the SERPINA1 gene or other DNA sequences that encoderegulatory elements of the SERPINA1 gene that results in permanentdeletion, insertion, or correction or modulation of one or moremutations or exons within or near the SERPINA1 gene or other DNAsequences that encode regulatory elements of the SERPINA1 gene, orwithin or near a safe harbor locus that results in permanent insertionof the SERPINA1 gene or minigene, and restoration of alpha-1-antitrypsin(AAT) protein activity.

In another method, Method 25, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19, or 24, wherein the one or moreDNA endonucleases is a Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7,Cas8, Cas9 (also known as Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3,Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1,Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16,CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease;or a homolog thereof, recombination of the naturally occurring molecule,codon-optimized, or modified version thereof, and combinations thereof.

In another method, Method 26, the present disclosure provides the methodof Method 25, wherein the method comprises introducing into the cell oneor more polynucleotides encoding the one or more DNA endonucleases.

In another method, Method 27, the present disclosure provides the methodof Method 25, wherein the method comprises introducing into the cell oneor more ribonucleic acids (RNAs) encoding the one or more DNAendonucleases.

In another method, Method 28, the present disclosure provides the methodof any one of Methods 26 or 27, wherein the one or more polynucleotidesor one or more RNAs is one or more modified polynucleotides or one ormore modified RNAs, the present disclosure provides the method of

In another method, Method 29, the present disclosure provides the methodof Method 26, wherein the DNA endonuclease is a protein or polypeptide.

In another method, Method 30, the present disclosure provides the methodof The method of any one of the preceding Methods, wherein the methodfurther comprises introducing into the cell one or more guideribonucleic acids (gRNAs).

In another method, Method 31, the present disclosure provides the methodof Method 30, wherein the one or more gRNAs are single-molecule guideRNA (sgRNAs).

In another method, Method 32, the present disclosure provides the methodof any one of Methods 30-31, wherein the one or more gRNAs or one ormore sgRNAs is one or more modified gRNAs or one or more modifiedsgRNAs.

In another method, Method 33, the present disclosure provides the methodof any one of Methods 30-32, wherein the one or more DNA endonucleasesis pre-complexed with one or more gRNAs or one or more sgRNAs.

In another method, Method 34, the present disclosure provides the methodof The method of any one of the preceding Methods, wherein the methodfurther comprises introducing into the cell a polynucleotide donortemplate comprising at least a portion of the wild-type SERPINA1 gene orminigene or cDNA.

In another method, Method 35, the present disclosure provides the methodof Method 34, wherein the at least a portion of the wild-type SERPINA1gene or minigene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5,intronic regions, fragments or combinations thereof, the entire SERPINA1gene, DNA sequences that encode wild type regulatory elements of theSERPINA1 gene, minigene or cDNA.

In another method, Method 36, the present disclosure provides the methodof any one of Methods 34-35, wherein the donor template is either asingle or double stranded polynucleotide.

In another method, Method 37, the present disclosure provides the methodof any one of Methods 34-36, wherein the donor template has homologousarms to the 14q32.13 region.

In another method, Method 38, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19, or 24, wherein the method furthercomprises introducing into the cell one guide ribonucleic acid (gRNA)and a polynucleotide donor template comprising at least a portion of thewild-type SERPINA1 gene, and wherein the one or more DNA endonucleasesis one or more Cas9 endonucleases that effect one double-strand break(DSB) at a DSB locus within or near the SERPINA1 gene or other DNAsequences that encode regulatory elements of the SERPINA1 gene, orwithin or near a safe harbor locus that facilitates insertion of a newsequence from the polynucleotide donor template into the chromosomal DNAat the locus or safe harbor locus that results in permanent insertion orcorrection of a part of the chromosomal DNA of the SERPINA1 gene orother DNA sequences that encode regulatory elements of the SERPINA1 geneproximal to the locus or safe harbor locus and restoration of AATprotein activity, and wherein the gRNA comprises a spacer sequence thatis complementary to a segment of the locus or safe harbor locus.

In another method, Method 39, the present disclosure provides the methodof Method 38, wherein proximal means nucleotides both upstream anddownstream of the locus or safe harbor locus.

In another method, Method 40, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19, or 24, wherein the method furthercomprises introducing into the cell two guide ribonucleic acid (gRNAs)and a polynucleotide donor template comprising at least a portion of thewild-type SERPINA1 gene, and wherein the one or more DNA endonucleasesis two or more Cas9 endonucleases that effect a pair of double-strandbreaks (DSBs), the first at a 5′ DSB locus and the second at a 3′ DSBlocus, within or near the SERPINA1 gene or other DNA sequences thatencode regulatory elements of the SERPINA1 gene, or within or near asafe harbor locus that facilitates insertion of a new sequence from thepolynucleotide donor template into the chromosomal DNA between the 5′DSB locus and the 3′ DSB locus that results in permanent insertion orcorrection of the chromosomal DNA between the 5′ DSB locus and the 3′DSB locus within or near the SERPINA1 gene or other DNA sequences thatencode regulatory elements of the SERPINA1 gene, or within or near asafe harbor locus and restoration of AAT protein activity, and whereinthe first guide RNA comprises a spacer sequence that is complementary toa segment of the 5′ DSB locus and the second guide RNA comprises aspacer sequence that is complementary to a segment of the 3′ DSB locus.

In another method, Method 41, the present disclosure provides the methodof any one of Methods 38-40, wherein the one or two gRNAs are one or twosingle-molecule guide RNA (sgRNAs).

In another method, Method 42, the present disclosure provides the methodof any one of Methods 38-41, wherein the one or two gRNAs or one or twosgRNAs is one or two modified gRNAs or one or two modified sgRNAs.

In another method, Method 43, the present disclosure provides the methodof any one of Methods 38-42, wherein the one or more DNA endonucleasesis pre-complexed with one or two gRNAs or one or two sgRNAs.

In another method, Method 44, the present disclosure provides the methodof any one of Methods 38-43, wherein the at least a portion of thewild-type SERPINA1 gene or minigene or cDNA is exon 1, exon 2, exon 3,exon 4, exon 5, intronic regions, fragments or combinations thereof, theentire SERPINA1 gene, DNA sequences that encode wild-type regulatoryelements of the SERPINA1 gene, minigene, or cDNA.

In another method, Method 45, the present disclosure provides the methodof any one of Methods 38-44, wherein the donor template is either asingle or double stranded polynucleotide.

In another method, Method 46, the present disclosure provides the methodof any one of Methods 38-45, wherein the donor template has homologousarms to the 14q32.13 region.

In another method, Method 47, the present disclosure provides the methodof Method 44, wherein the DSB, or 5′ DSB and 3′ DSB are in the first,second, third, fourth, fifth exon or intron, SERPINA1 gene.

In another method, Method 48, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19, 24, 30-33, or 38-41, wherein thegRNA or sgRNA is directed to one or more of the following pathologicalvariants: rs764325655, rs121912713, rs28929474, rs17580, rs121912714,rs764220898, rs199422211, rs751235320, rs199422210, rs267606950,rs55819880, rs28931570.

In another method, Method 49, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19, or 24-48, wherein the insertionor correction is by homology directed repair (HDR).

In another method, Method 50, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19, or 24, wherein the method furthercomprises introducing into the cell two guide ribonucleic acid (gRNAs),and wherein the one or more DNA endonucleases is two or more Cas9endonucleases that effect a pair of double-strand breaks (DSBs), thefirst at a 5′ DSB locus and the second at a 3′ DSB locus, within or nearthe SERPINA1 gene that causes a deletion of the chromosomal DNA betweenthe 5′ DSB locus and the 3′ DSB locus that results in permanent deletionof the chromosomal DNA between the 5′ DSB locus and the 3′ DSB locuswithin or near the SERPINA1 gene and restoration of AAT proteinactivity, and wherein the first guide RNA comprises a spacer sequencethat is complementary to a segment of the 5′ DSB locus and the secondguide RNA comprises a spacer sequence that is complementary to a segmentof the 3′ DSB locus.

In another method, Method 51, the present disclosure provides the methodof Method 50, wherein the two gRNAs are two single-molecule guide RNA(sgRNAs).

In another method, Method 52, the present disclosure provides the methodof any one of Methods 50-51, wherein the two gRNAs or two sgRNAs are twomodified gRNAs or two modified sgRNAs.

In another method, Method 53, the present disclosure provides the methodof any one of Methods 50-52, wherein the one or more DNA endonucleasesis pre-complexed with one or two gRNAs or one or two sgRNAs.

In another method, Method 54, the present disclosure provides the methodof any one of Methods 50-53, wherein both the 5′ DSB and 3′ DSB are inor near either the first exon, second exon, third exon, fourth exon orfifth exon of the SERPINA1 gene.

In another method, Method 55, the present disclosure provides the methodof any one of Method 50-54, wherein the deletion is a deletion of 1 kbor less.

In another method, Method 56, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, or 19-55, wherein the Cas9 or Cpf1mRNA, gRNA, and a donor template are either each formulated intoseparate lipid nanoparticles or all co-formulated into a lipidnanoparticle.

In another method, Method 57, the present disclosure provides the methodof any one of Methods 1, 6, 11, 15 or 19-55, wherein the Cas9 or Cpf1mRNA is formulated into a lipid nanoparticle, and both the gRNA anddonor template are delivered by a viral vector.

In another method, Method 58, the present disclosure provides the methodof Method 57, wherein the viral vector is an adeno-associated virus(AAV).

In another method, Method 59, the present disclosure provides the methodof Method 58, wherein the AAV vector is an AAV6 vector.

In another method, Method 60, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19 or 24-55, wherein the Cas9 or Cpf1mRNA, gRNA and a donor template are either each formulated into separateexosomes or all co-formulated into an exosome.

In another method, Method 61, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19 or 24-55, wherein the Cas9 or Cpf1mRNA is formulated into a lipid nanoparticle, and the gRNA is deliveredto the cell by electroporation and donor template is delivered to thecell by a viral vector, the present disclosure provides the method of

In another method, Method 62, the present disclosure provides the methodof Method 61, wherein the viral vector is an adeno-associated virus(AAV) vector.

In another method, Method 63, the present disclosure provides the methodof Method 62, wherein the AAV vector is an AAV6 vector.

In another method, Method 64, the present disclosure provides the methodof any one of the preceding Methods, wherein the SERPINA1 gene islocated on Chromosome 14: 1,493,319-1,507,264 (Genome ReferenceConsortium—GRCh38/hg38).

In another method, Method 65, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19 or 24-55, wherein the restorationof AAT protein activity is compared to wild-type or normal AAT proteinactivity.

In another method, Method 66, the present disclosure provides the methodof Method 1, wherein the human cell is a liver cell.

In another method, Method 67, the present disclosure provides the methodof Method 23, wherein the cell is a liver cell.

In another method, Method 68, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19 or 24-55, wherein the SERPINA1gene is operably linked to an exogenous promoter that drives expressionof the SERPINA1 gene.

In another method, Method 69, the present disclosure provides the methodof any one of Methods 1, 2, 7, 13, 19 or 24-55, wherein the one or moreDSBs occurs at a location immediately 3′ to an endogenous promoterlocus.

The present disclosure also provides a composition, Composition 1, ofone or more guide ribonucleic acids (gRNAs) for editing a SERPINA1 genein a cell from a patient with alpha-1 antitrypsin deficiency (AATD), theone or more gRNAs comprising a spacer sequence selected from the groupconsisting of the nucleic acid sequences in SEQ ID NOs: 54,860-68,297for editing the SERPINA1 gene in a cell from a patient with alpha-1antitrypsin deficiency.

In another composition, Composition 2, the present disclosure providesthe composition of Composition 1, wherein the one or more gRNAs are oneor more single-molecule guide RNAs (sgRNAs).

In another composition, Composition 3, the present disclosure providesthe composition of Composition 1 or Composition 2, wherein the one ormore gRNAs or one or more sgRNAs is one or more modified gRNAs or one ormore modified sgRNAs.

In another composition, Composition 4, the present disclosure providesthe composition of Composition 1 or Composition 2 or Composition 3,wherein the cell is a liver cell.

The present disclosure also provides a composition, Composition 5, ofone or more guide ribonucleic acids (gRNAs) for editing a safe harborlocus in a cell from a patient with alpha-1 antitrypsin deficiency(AATD), the one or more gRNAs comprising a spacer sequence selected fromthe group consisting of the nucleic acid sequences in SEQ ID NOs:1-54,859 for editing the safe harbor locus in a cell from a patient withalpha-1 antitrypsin deficiency, wherein the safe harbor locus isselected from the group consisting of AAVS1 (PPP1R12C), ALB, Angptl3,ApoC3, ASGR2, CCR5, FIX (F9), G6PC, Gys2, HGD, Lp(a), Pcsk9, Serpina1,TF, and TTR.

In another composition, Composition 6, the present disclosure providesthe composition of Composition 5, wherein the one or more gRNAs are oneor more single-molecule guide RNAs (sgRNAs).

In another composition, Composition 7, the present disclosure providesthe composition of Composition 5 or Composition 6, wherein the one ormore gRNAs or one or more sgRNAs is one or more modified gRNAs or one ormore modified sgRNAs.

In another composition, Composition 8, the present disclosure providesthe composition of Composition 5 or Composition 6 or Composition 7,wherein the cell is a liver cell.

Definitions

The term “comprising” or “comprises” is used in reference tocompositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The term “consisting essentially of” refers to those elements requiredfor a given embodiment. The term permits the presence of additionalelements that do not materially affect the basic and novel or functionalcharacteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

The singular forms “a,” “an,” and “the” include plural references,unless the context clearly dictates otherwise.

Certain numerical values presented herein are preceded by the term“about.” The term “about” is used to provide literal support for thenumerical value the term “about” precedes, as well as a numerical valuethat is approximately the numerical value, that is the approximatingunrecited numerical value may be a number which, in the context it ispresented, is the substantial equivalent of the specifically recitednumerical value. The term “about” means numerical values within ±10% ofthe recited numerical value.

When a range of numerical values is presented herein, it is contemplatedthat each intervening value between the lower and upper limit of therange, the values that are the upper and lower limits of the range, andall stated values with the range are encompassed within the disclosure.All the possible sub-ranges within the lower and upper limits of therange are also contemplated by the disclosure.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples, which provide illustrative non-limiting embodimentsof the invention.

The examples describe the use of the CRISPR system as an illustrativegenome editing technique to create defined therapeutic genomicreplacements, termed “genomic modifications” herein, in the SERPINA1gene that lead to permanent correction of mutations in the genomiclocus, or expression at a heterologous locus, that restore AAT proteinactivity. Introduction of the defined therapeutic modificationsrepresents a novel therapeutic strategy for the potential ameliorationof AATD, as described and illustrated herein.

Example 1—CRISPR/SpCas9 Target Sites for the SERPINA1 Gene (Other thanas a Safe Harbor Locus)

Regions of the SERPINA1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNRG. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 54,860-61,324.

Example 2—CRISPR/SaCas9 Target Sites for the SERPINA1 Gene (Other thanas a Safe Harbor Locus)

Regions of the SERPINA1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 61,324-61,936.

Example 3—CRISPR/StCas9 Target Sites for the SERPINA1 Gene (Other thanas a Safe Harbor Locus)

Regions of the SERPINA1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 61,937-62,069.

Example 4—CRISPR/TdCas9 Target Sites for the SERPINA1 Gene (Other thanas a Safe Harbor Locus)

Regions of the SERPINA1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 69,070-62,120.

Example 5—CRISPR/NmCas9 Target Sites for the SERPINA1 Gene (Other thanas a Safe Harbor Locus)

Regions of the SERPINA1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceNNNNGHTT. gRNA 20 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 62,121-62,563.

Example 6—CRISPR/Cpf1 Target Sites for the SERPINA1 Gene (Other than asa Safe Harbor Locus)

Regions of the SERPINA1 gene were scanned for target sites. Each areawas scanned for a protospacer adjacent motif (PAM) having the sequenceYTN. gRNA 22 bp spacer sequences corresponding to the PAM wereidentified, as shown in SEQ ID NOs: 62,564-68,297.

Example 7—CRISPR/SpCas9 Target Sites for Safe Harbor Loci

The following safe harbor loci were scanned for target sites: Exons 1-2of AAVS1 (PPP1R12C), Exons 1-2 of ALB, Exons 1-2 of Angptl3, Exons 1-2of ApoC3, Exons 1-2 of ASGR2, Exons 1-2 of CCR5, Exons 1-2 of FIX (F9),Exons 1-2 of G6PC, Exons 1-2 of Gys2, Exons 1-2 of HGD, Exons 1-2 ofLp(a), Exons 1-2 of Pcsk9, Exons 1-2 of Serpina1, Exons 1-2 of TF, andExons 1-2 of TTR. Each area was scanned for a protospacer adjacent motif(PAM) having the sequence NRG. gRNA 20 bp spacer sequences correspondingto the PAM were identified, as shown in the following sequences: AAVS1(PPP1R12C): SEQ ID NOs. 1-2,032; ALB: SEQ ID NOs. 3,482-3,649; Angptl3:SEQ ID NOs. 4,104-4,448; ApoC3: SEQ ID NOs. 5,432-5,834; ASGR2: SEQ IDNOs. 6,109-7,876; CCR5: SEQ ID NOs. 9,642-9,844; FIX (F9): SEQ ID NOs.10,221-11,686; G6PC: SEQ ID NOs. 14,230-15,245; Gys2: SEQ ID NOs.16,581-22,073; HGD: SEQ ID NOs. 32,254-33,946; Lp(a): SEQ ID NOs.36,789-40,583; Pcsk9: SEQ ID NOs. 46,154-48,173; Serpina1: SEQ ID NOs.50,345-51,482; TF: SEQ ID NOs. 52,446-53,277; and TTR: SEQ ID NOs.54,063-54,362.

Example 8—CRISPR/SaCas9 Target Sites for Safe Harbor Loci

The following safe harbor loci were scanned for target sites: Exons 1-2of AAVS1 (PPP1R12C), Exons 1-2 of ALB, Exons 1-2 of Angptl3, Exons 1-2of ApoC3, Exons 1-2 of ASGR2, Exons 1-2 of CCR5, Exons 1-2 of FIX (F9),Exons 1-2 of G6PC, Exons 1-2 of Gys2, Exons 1-2 of HGD, Exons 1-2 ofLp(a), Exons 1-2 of Pcsk9, Exons 1-2 of Serpina1, Exons 1-2 of TF, andExons 1-2 of TTR. Each area was scanned for a protospacer adjacent motif(PAM) having the sequence NNGRRT. gRNA 20 bp spacer sequencescorresponding to the PAM were identified, as shown in the followingsequences: AAVS1 (PPP1R12C): SEQ ID NOs. 2,033-2,203; ALB: SEQ ID NOs.3,650-3,677; Angptl3: SEQ ID NOs. 4,449-4,484; ApoC3: SEQ ID NOs.5,835-5,859; ASGR2: SEQ ID NOs. 7,877-8,082; CCR5: SEQ ID NOs.9,845-9,876; FIX (F9): SEQ ID NOs. 11,687-11,849; G6PC: SEQ ID NOs.15,246-15,362; Gys2: SEQ ID NOs. 22,074-22,749; HGD: SEQ ID NOs.33,947-34,160; Lp(a): SEQ ID NOs. 40,584-40,993; Pcsk9: SEQ ID NOs.48,174-48,360; Serpina1: SEQ ID NOs. 51,483-51,575; TF: SEQ ID NOs.53,278-53,363; and TTR: SEQ ID NOs. 54,363-54,403.

Example 9—CRISPR/StCas9 Target Sites for Safe Harbor Loci

The following safe harbor loci were scanned for target sites: Exons 1-2of AAVS1 (PPP1R12C), Exons 1-2 of ALB, Exons 1-2 of Angptl3, Exons 1-2of ApoC3, Exons 1-2 of ASGR2, Exons 1-2 of CCR5, Exons 1-2 of FIX (F9),Exons 1-2 of G6PC, Exons 1-2 of Gys2, Exons 1-2 of HGD, Exons 1-2 ofLp(a), Exons 1-2 of Pcsk9, Exons 1-2 of Serpina1, Exons 1-2 of TF, andExons 1-2 of TTR. Each area was scanned for a protospacer adjacent motif(PAM) having the sequence NNAGAAW. gRNA 20 bp spacer sequencescorresponding to the PAM were identified, as shown in the followingsequences: AAVS1 (PPP1R12C): SEQ ID NOs. 2,204-2,221; ALB: SEQ ID NOs.3,678-3,695; Angptl3: SEQ ID NOs. 4,485-4,507; ApoC3: SEQ ID NOs.5,860-5,862; ASGR2: SEQ ID NOs. 8,083-8,106; CCR5: SEQ ID NOs.9,877-9,890; FIX (F9): SEQ ID NOs. 11,850-11,910; G6PC: SEQ ID NOs.15,363-15,386; Gys2: SEQ ID NOs. 22,750-20,327; HGD: SEQ ID NOs.34,161-34,243; Lp(a): SEQ ID NOs. 40,994-41,129; Pcsk9: SEQ ID NOs.48,361-48,396; Serpina1: SEQ ID NOs. 51,576-51,587; TF: SEQ ID NOs.53,364-53,375; and TTR: SEQ ID NOs. 54,404-54,420.

Example 10—CRISPR/TdCas9 Target Sites for Safe Harbor Loci

The following safe harbor loci were scanned for target sites: Exons 1-2of AAVS1 (PPP1R12C), Exons 1-2 of ALB, Exons 1-2 of Angptl3, Exons 1-2of ApoC3, Exons 1-2 of ASGR2, Exons 1-2 of CCR5, Exons 1-2 of FIX (F9),Exons 1-2 of G6PC, Exons 1-2 of Gys2, Exons 1-2 of HGD, Exons 1-2 ofLp(a), Exons 1-2 of Pcsk9, Exons 1-2 of Serpina1, Exons 1-2 of TF, andExons 1-2 of TTR. Each area was scanned for a protospacer adjacent motif(PAM) having the sequence NAAAAC. gRNA 20 bp spacer sequencescorresponding to the PAM were identified, as shown in the followingsequences: AAVS1 (PPP1R12C): SEQ ID NOs. 2,222-2,230; ALB: SEQ ID NOs.3,696-3,700; Angptl3: SEQ ID NOs. 4,508-4,520; ApoC3: SEQ ID NOs.5,863-5,864; ASGR2: SEQ ID NOs. 8,107-8,118; CCR5: SEQ ID NOs.9,891-9,892; FIX (F9): SEQ ID NOs. 11,911-11,935; G6PC: SEQ ID NOs.15,387-15,395; Gys2: SEQ ID NOs. 23,028-23,141; HGD: SEQ ID NOs.34,244-34,262; Lp(a): SEQ ID NOs. 41,130-41,164; Pcsk9: SEQ ID NOs.48,397-48,410; Serpina1: SEQ ID NOs. 51,588-51,590; TF: SEQ ID NOs.53,376-53,382; and TTR: SEQ ID NOs. 54,421-54,422.

Example 11—CRISPR/NmCas9 Target Sites for Safe Harbor Loci

The following safe harbor loci were scanned for target sites: Exons 1-2of AAVS1 (PPP1R12C), Exons 1-2 of ALB, Exons 1-2 of Angptl3, Exons 1-2of ApoC3, Exons 1-2 of ASGR2, Exons 1-2 of CCR5, Exons 1-2 of FIX (F9),Exons 1-2 of G6PC, Exons 1-2 of Gys2, Exons 1-2 of HGD, Exons 1-2 ofLp(a), Exons 1-2 of Pcsk9, Exons 1-2 of Serpina1, Exons 1-2 of TF, andExons 1-2 of TTR. Each area was scanned for a protospacer adjacent motif(PAM) having the sequence NNNNGHTT. gRNA 20 bp spacer sequencescorresponding to the PAM were identified, as shown in the followingsequences: AAVS1 (PPP1R12C): SEQ ID NOs. 2,231-2,305; ALB: SEQ ID NOs.3,701-3,724; Angptl3: SEQ ID NOs. 4,521-4,583; ApoC3: SEQ ID NOs.5,865-5,876; ASGR2: SEQ ID NOs. 8,119-8,201; CCR5: SEQ ID NOs.9,893-9,920; FIX (F9): SEQ ID NOs. 11,936-12,088; G6PC: SEQ ID NOs.15,396-15,485; Gys2: SEQ ID NOs. 23,142-23,821; HGD: SEQ ID NOs.34,263-34,463; Lp(a): SEQ ID NOs. 41,165-41,532; Pcsk9: SEQ ID NOs.48,411-48,550; Serpina1: SEQ ID NOs. 51,591-51,641; TF: SEQ ID NOs.53,383-53,426; and TTR: SEQ ID NOs. 54,423-54,457.

Example 12—CRISPR/Cpf1 Target Sites for Safe Harbor Loci

Exons 1-2 of the AAVS1 (PPP1R12C) gene were scanned for target sites.The following safe harbor loci were scanned for target sites: Exons 1-2of AAVS1 (PPP1R12C), Exons 1-2 of ALB, Exons 1-2 of Angptl3, Exons 1-2of ApoC3, Exons 1-2 of ASGR2, Exons 1-2 of CCR5, Exons 1-2 of FIX (F9),Exons 1-2 of G6PC, Exons 1-2 of Gys2, Exons 1-2 of HGD, Exons 1-2 ofLp(a), Exons 1-2 of Pcsk9, Exons 1-2 of Serpina1, Exons 1-2 of TF, andExons 1-2 of TTR. Each area was scanned for a protospacer adjacent motif(PAM) having the sequence YTN. gRNA 22 bp spacer sequences correspondingto the PAM were identified, as shown in the following sequences: AAVS1(PPP1R12C): SEQ ID NOs. 2,306-3,481; ALB: SEQ ID NOs. 3,725-4,103;Angptl3: SEQ ID NOs. 4,584-5,431; ApoC3: SEQ ID NOs. 5,877-6,108; ASGR2:SEQ ID NOs. 8,202-9,641; CCR5: SEQ ID NOs. 9,921-10,220; FIX (F9): SEQID NOs. 12,089-14,229; G6PC: SEQ ID NOs. 15,486-16,580; Gys2: SEQ IDNOs. 23,822-32,253; HGD: SEQ ID NOs. 34,464-36,788; Lp(a): SEQ ID NOs.41,533-46,153; Pcsk9: SEQ ID NOs. 48,551-50,344; Serpina1: SEQ ID NOs.51,642-52,445; TF: SEQ ID NOs. 53,427-54,062; and TTR: SEQ ID NOs.54,458-54,859.

Example 13—Bioinformatics Analysis of the Guide Strands

Candidate guides will be screened and selected in a multi-step processthat involves both theoretical binding and experimentally assessedactivity. By way of illustration, candidate guides having sequences thatmatch a particular on-target site, such as a site within the SERPINA1gene, with adjacent PAM can be assessed for their potential to cleave atoff-target sites having similar sequences, using one or more of avariety of bioinformatics tools available for assessing off-targetbinding, as described and illustrated in more detail below, in order toassess the likelihood of effects at chromosomal positions other thanthose intended. Candidates predicted to have relatively lower potentialfor off-target activity can then be assessed experimentally to measuretheir on-target activity, and then off-target activities at varioussites. Preferred guides have sufficiently high on-target activity toachieve desired levels of gene editing at the selected locus, andrelatively lower off-target activity to reduce the likelihood ofalterations at other chromosomal loci. The ratio of on-target tooff-target activity is often referred to as the “specificity” of aguide.

For initial screening of predicted off-target activities, there are anumber of bioinformatics tools known and publicly available that can beused to predict the most likely off-target sites; and since binding totarget sites in the CRISPR/Cas9/Cpf1 nuclease system is driven byWatson-Crick base pairing between complementary sequences, the degree ofdissimilarity (and therefore reduced potential for off-target binding)is essentially related to primary sequence differences: mismatches andbulges, i.e. bases that are changed to a non-complementary base, andinsertions or deletions of bases in the potential off-target siterelative to the target site. An exemplary bioinformatics tool calledCOSMID (CRISPR Off-target Sites with Mismatches, Insertions andDeletions) (available on the web at crispr.bme.gatech.edu) compiles suchsimilarities. Other bioinformatics tools include, but are not limitedto, GUIDO, autoCOSMID, and CCtop.

Bioinformatics were used to minimize off-target cleavage in order toreduce the detrimental effects of mutations and chromosomalrearrangements. Studies on CRISPR/Cas9 systems suggested the possibilityof high off-target activity due to nonspecific hybridization of theguide strand to DNA sequences with base pair mismatches and/or bulges,particularly at positions distal from the PAM region. Therefore, it isimportant to have a bioinformatics tool that can identify potentialoff-target sites that have insertions and/or deletions between the RNAguide strand and genomic sequences, in addition to base-pair mismatches.The bioinformatics-based tool, COSMID (CRISPR Off-target Sites withMismatches, Insertions and Deletions) was therefore used to searchgenomes for potential CRISPR off-target sites (available on the web atcrispr.bme.gatech.edu). COSMID output ranked lists of the potentialoff-target sites based on the number and location of mismatches,allowing more informed choice of target sites, and avoiding the use ofsites with more likely off-target cleavage.

Additional bioinformatics pipelines were employed that weigh theestimated on- and/or off-target activity of gRNA targeting sites in aregion. Other features that may be used to predict activity includeinformation about the cell type in question, DNA accessibility,chromatin state, transcription factor binding sites, transcriptionfactor binding data, and other CHIP-seq data. Additional factors areweighed that predict editing efficiency, such as relative positions anddirections of pairs of gRNAs, local sequence features andmicro-homologies.

Example 14—Testing of Preferred Guides in Cells for On-Target Activity

The gRNAs predicted to have the lowest off target activity will then betested for on-target activity in a model cell line, such as Huh-7 cells,and evaluated for indel frequency using TIDE or next generationsequencing. TIDE is a web tool to rapidly assess genome editing byCRISPR-Cas9 of a target locus determined by a guide RNA (gRNA or sgRNA).Based on the quantitative sequence trace data from two standardcapillary sequencing reactions, the TIDE software quantifies the editingefficacy and identifies the predominant types of insertions anddeletions (indels) in the DNA of a targeted cell pool. See Brinkman etal, Nucl. Acids Res. (2014) for a detailed explanation and examples.Next-generation sequencing (NGS), also known as high-throughputsequencing, is the catch-all term used to describe a number of differentmodern sequencing technologies including: Illumina (Solexa) sequencing,Roche 454 sequencing, Ion torrent: Proton/PGM sequencing, and SOLiDsequencing. These recent technologies allow one to sequence DNA and RNAmuch more quickly and cheaply than the previously used Sangersequencing, and as such have revolutionized the study of genomics andmolecular biology.

Transfection of tissue culture cells, allows screening of differentconstructs and a robust means of testing activity and specificity.Tissue culture cell lines, such as Huh-7 cells, are easily transfectedand result in high activity. These or other cell lines will be evaluatedto determine the cell lines that provide the best surrogate. These cellswill then be used for many early stage tests. For example, individualgRNAs for S. pyogenes Cas9 will be transfected into the cells usingplasmids, such as, for example, CTx-1, CTx-2, or CTx-3 described in FIG.1A-1C, which are suitable for expression in human cells. Several dayslater, the genomic DNA is harvested and the target site amplified byPCR. The cutting activity can be measured by the rate of insertions,deletions and mutations introduced by NHEJ repair of the free DNA ends.Although this method cannot differentiate correctly repaired sequencesfrom uncleaved DNA, the level of cutting can be gauged by the amount ofmis-repair. Off-target activity can be observed by amplifying identifiedputative off-target sites and using similar methods to detect cleavage.Translocation can also be assayed using primers flanking cut sites, todetermine if specific cutting and translocations happen. Un-guidedassays have been developed allowing complementary testing of off-targetcleavage including guide-seq. The gRNA or pairs of gRNA with significantactivity can then be followed up in cultured cells to measure correctionof SERPINA1 mutation. Off-target events can be followed again. Theseexperiments allow optimization of nuclease and donor design anddelivery.

Example 15—Testing of Preferred Guides in Cells for Off-Target Activity

The gRNAs having the best on-target activity from the TIDE and nextgeneration sequencing studies in the above example will then be testedfor off-target activity using whole genome sequencing.

Example 16—Testing Different Approaches for HDR Gene Editing

After testing the gRNAs for both on-target activity and off-targetactivity, the mutation correction and knock-in strategies will be testedfor HDR gene editing.

For the mutation correction approach, the donor DNA template will beprovided as a short single-stranded oligonucleotide, a shortdouble-stranded oligonucleotide (PAM sequence intact/PAM sequencemutated), a long single-stranded DNA molecule (PAM sequence intact/PAMsequence mutated) or a long double-stranded DNA molecule (PAM sequenceintact/PAM sequence mutated). In addition, the donor DNA template willbe delivered by AAV.

For the cDNA knock-in approach, a single-stranded or double-stranded DNAhaving homologous arms to the 14q32.13 region may include more than 40nt of the first exon (the first coding exon) of the SERPINA1 gene, thecomplete CDS of the SERPINA1 gene and 3′UTR of the SERPINA1 gene, and atleast 40 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the 14q32.13 region, whichincludes more than 80 nt of the first exon of the SERPINA1 gene, thecomplete CDS of the SERPINA1 gene and 3′UTR of the SERPINA1 gene, and atleast 80 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the 14q32.13 region mayinclude more than 100 nt of the first exon of the SERPINA1 gene, thecomplete CDS of the SERPINA1 gene and 3′UTR of the SERPINA1 gene, and atleast 100 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the 14q32.13 region mayinclude more than 150 nt of the first exon of the SERPINA1 gene, thecomplete CDS of the SERPINA1 gene and 3′UTR of the SERPINA1 gene, and atleast 150 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the 14q32.13 region mayinclude more than 300 nt of the first exon of the SERPINA1 gene, thecomplete CDS of the SERPINA1 gene and 3′UTR of the SERPINA1 gene, and atleast 300 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the 14q32.13 region mayinclude more than 400 nt of the first exon of the SERPINA1 gene, thecomplete CDS of the SERPINA1 gene and 3′UTR of the SERPINA1 gene, and atleast 400 nt of the following the first intron. Alternatively, the DNAtemplate will be delivered by AAV.

For the cDNA or minigene knock-in approach, a single-stranded ordouble-stranded DNA having homologous arms to the 14q32.13, whichincludes more than 80 nt of the second exon (the first coding exon) ofthe SERPINA1 gene, the complete CDS of the SERPINA1 gene and 3′UTR ofthe SERPINA1 gene, and at least 80 nt of the following intron.Alternatively, the DNA template will be delivered by AAV.

Next, the efficiency of HDR mediated correction of the common mutationof the PI Z allele of SERPINA1 and knock-in of cDNA minigene (comprisedof, natural or synthetic enhancer and promoter, one or more exons, andnatural or synthetic introns, and natural or synthetic 3′UTR andpolyadenylation signal) into the 2^(nd) exon will be assessed.

Example 17—Re-Assessment of Lead CRISPR-Cas9/DNA Donor Combinations

After testing the different strategies for HDR gene editing, the leadCRISPR-Cas9/DNA donor combinations will be re-assessed in primary humanhepatocytes for efficiency of deletion, recombination, and off-targetspecificity. Cas9 mRNA or RNP will be formulated into lipidnanoparticles for delivery, sgRNAs will be formulated into nanoparticlesor delivered as AAV, and donor DNA will be formulated into nanoparticlesor delivered as AAV.

Example 18—In Vivo Testing in Relevant Mouse Model

After the CRISPR-Cas9/DNA donor combinations have been re-assessed, thelead formulations will be tested in vivo in an animal model. Suitableanimal models include, by way of non-limiting example, a FGR mouse modelwith the livers repopulated with human hepatocytes or iPSC derivedhepatocytes (normal or AAT deficient).

Culture in human cells allows direct testing on the human target and thebackground human genome, as described above.

Preclinical efficacy and safety evaluations can be observed throughengraftment of modified mouse or human hepatocytes in FGR mice. Themodified cells can be observed in the months after engraftment.

Example 19—Screening of gRNAs

To identify a large spectrum of pairs of gRNAs able to edit the SERPINA1DNA target region, an in vitro transcribed (IVT) gRNA screen wasconducted. SERPINA1 genomic sequence was submitted for analysis usinggRNA design software. The resulting list of gRNAs was narrowed to a listof about 200 gRNAs based on uniqueness of sequence—only gRNAs without aperfect match somewhere else in the genome were screened—and minimalpredicted off targets. This set of gRNAs was transcribed in vitro andtransfected using messenger Max into HEK293T cells that constitutivelyexpress Cas9. Cells were harvested 48 hours post transfection, thegenomic DNA was isolated, and cutting efficiency was evaluated usingTIDE analysis. The cutting efficiency of the gRNA (% indels) is listedin Table 4.

It was found that about 14% of the tested gRNAs induced cuttingefficiencies over 70%. (FIG. 3 ).

TABLE 4 gRNA sequences and cutting efficiencies in HEK293T-Cas9 cellsGuide Name Guide Sequence % Indel SERPINA1_T61CCCAATGTCTAGAAGGTCTT (SEQ ID NO: 55, 783) 70.3 SFRPINA1_T37GCCCAATGTCTAGAAGGTCT (SEQ ID NO: 55, 782) 50.6 SERPINA1_T185ATCAAGGCCCAATGTCTAGA (SEQ ID NO: 55, 781) 58.1 SERPINA1_T70GCCCAAGACCTTCTAGACAT (SEQ ID NO: 60, 476) 56.1 SERPINA1_T88CCCAAGACCTTCTAGACATT (SEQ ID NO: 60, 477) 60 SERPINA1_T162GTGTCAGTATGGATAAATCA (SEQ ID NO: 55, 778) 59.2 SFRPINA1_T170CAAACCTTTCTGTGTCAGTA (SEQ ID NO: 55, 776) 7.1 SERPINA1_T390TTATCCATACTGACACAGAA (SEQ ID NO: 60, 480) 64.7 SERPINA1_T388CATACTGACACAGAAAGGTT (SEQ ID NO: 60, 481) 63.6 SERPINA1_T283ATACTGACACAGAAAGGTTT (SEQ ID NO: 60, 482) 70.5 SERPINA1_T282TTTGGGCTAAGTTGTTTCAA (SEQ ID NO: 60, 485) 40.5 SERPINA1_T72CACCAAATCTCACAGATCGA (SEQ ID NO: 55, 771) 81.6 SFRPINA1_T73CTCCTTCGATCTGTGAGATT (SEQ ID NO: 60, 487) 47.3 SERPINA1_T259GCTAAAGATGACACTTATTT (SEQ ID NO: 60, 490) 73.9 SERPINA1_T275CACTTATTTTGGAAAACTAA (SEQ ID NO: 60, 492) 0.6 SERPINA1_T182ATGGAACTGCAGTTGTTCAT (SEQ ID NO: 55, 764) 52.8 SERPINA1_T323AACAACTGCAGTTCCATGAA (SEQ ID NO: 60, 494) 66.2 SERPINA1_T383CAAGATAATGCAGCCATTCA (SEQ ID NO: 55, 762) 50.7 SERPINA1_T177CATGAATGGCTGCATTATCT (SEQ ID NO: 60, 495) 22 SERPINA1_T280ATGAATGGCTGCATTATCTT (SEQ ID NO: 60, 496) 57.8 SERPINA1_T244TGAATGGCTGCATTATCTTG (SEQ ID NO: 60, 497) 51.8 SERPINA1_T66GGCTGCATTATCTTGGGGTC (SEQ ID NO: 60, 498) 18 SERPINA1_T128GCTGCATTATCTTGGGGTCT (SEQ ID NO: 60, 499) 34.8 5FRPINA1_T385CACTGTGAAGGTCACTGCCA (SEQ ID NO: 60, 504) 50.8 SERPINA1_T194CTCCTTGAGGACACGGACCC (SEQ ID NO: 55, 756) 15.2 SERPINA1_T418TGCCAGGGTCCGTGTCCTCA (SEQ ID NO: 60, 506) 49.6 SERPINA1_T59ACACGGCTTGAAGCTCCTTG (SEQ ID NO: 55, 754) 49.2 SERPINA1_T257GCTCTCTCCTTTCTAGTACA (SEQ ID NO: 55, 751) 90.1 SERPINA1_T13CTTCAAGCCGTGTACTAGAA (SEQ ID NO: 60, 511) 15.5 SERPINA1_T291TACTAGAAAGGAGAGAGCCC (SEQ ID NO: 60, 515) 45.8 SERPINA1_T431TAGAAAGGAGAGAGCCCTGG (SEQ ID NO: 60, 517) 60.1 SERPINA1_T341TCACTCCACGTCTGCCTCCA (SEQ ID NO: 55, 749) 83.9 SERPINA1_T471GTCACTCCACGTCTGCCTCC (SEQ ID NO: 55, 748) 51.7 SERPINA1_T294GAGAGCCCTGGAGGCAGACG (SEQ ID NO: 60, 519) 44.8 SERPINA1_T187TGCACCCACAACTCAGAACA (SEQ ID NO: 55, 744) 77.1 SERPINA1_T92GTGCACCCACAACTCAGAAC (SEQ ID NO: 55, 743) 46.5 SERPINA1_T314CTCTTCCCTGTTCTGAGTTG (SEQ ID NO: 60, 522) 27.8 SERPINA1_T120AGTTGTGGGTGCACCTGAGC (SEQ ID NO: 60, 526) 10.6 SERPINA1_T224GTTGTGGGTGCACCTGAGCA (SEQ ID NO: 60, 527) 72.5 SERPINA1_T274TTGTGGGTGCACCTGAGCAG (SEQ ID NO: 60, 528) 39.8 SERPINA1_T167AAGCGCCTCTCCCCCTGCTC (SEQ ID NO: 55, 740) 36.8 SERPINA1_T437TGTGGGTGCACCTGAGCAGG (SEQ ID NO: 60, 529) 42.7 SERPINA1_T81CAGGGGGAGAGGCGCTTGTC (SEQ ID NO: 60, 534) 15.8 SERPINA1_T168AGAGGCGCTTGTCAGGAAGA (SEQ ID NO: 60, 536) 38 SERPINA1_T336CTTGTCAGGAAGATGGACAG (SEQ ID NO: 60, 539) 46 SERPINA1_T360TTGTCAGGAAGATGGACAGA (SEQ ID NO: 60, 540) 45.1 SERPINA1_T405TGTCAGGAAGATGGACAGAG (SEQ ID NO: 60, 541) 80.2 SERPINA1_T379AAGGCTTTGGCTGATGGGGC (SEQ ID NO: 55, 737) 17.5 SERPINA1_T180CCTCAAGGCTTTGGCTGATG (SEQ ID NO: 55, 736) 14 SERPINA1_T135TCCTCAAGGCTTTGGCTGAT (SEQ ID NO: 55, 735) 25.9 SERPINA1_T197CTCCTCAAGGCTTTGGCTGA (SEQ ID NO: 55, 734) 20.3 SERPINA1_T345CTTGCTCCTCCTCAAGGCTT (SEQ ID NO: 55, 733) 23.8 SERPIN20_T150CCCCATCAGCCAAAGCCTTG (SEQ ID NO: 60, 547) 34.9 SERPINA1_T328CATCAGCCAAAGCCTTGAGG (SEQ ID NO: 60, 549) 35.7 SERPINA1_T138ATAGGCCTTGCTCCTCCTCA (SEQ ID NO: 55, 732) 76.2 SERPINA1_T348CAAAGCCTTGAGGAGGAGCA (SEQ ID NO: 60, 552) 24.4 SERPINA1_T176AGGAGCAAGGCCTATGTGAC (SEQ ID NO: 60, 554) 32.7 SERPINA1_T376CCTCTCCCTCCCTGTCACAT (SEQ ID NO: 55, 730) 70.1 SERPINA1_T308GGAGCAAGGCCTATGTGACA (SEQ ID NO: 60, 555) 58.8 SERPINA1_T181GCAAGGCCTATGTGACAGGG (SEQ ID NO: 60, 557) 71 SERPINA1_T265CAAGGCCTATGTGACAGGGA (SEQ ID NO: 60, 558) 44.6 SERPINA1_T465CCTATGTGACAGGGAGGGAG (SEQ ID NO: 60, 561) 24.5 SERPINA1_T392CAGGGAGGGAGAGGATGTGC (SEQ ID NO: 60, 563) 26.9 SERPINA1_T372AGGGAGGGAGAGGATGTGCA (SEQ ID NO: 60, 564) 74.3 SERPINA1_T459GGGAGAGGATGTGCAGGGCC (SEQ ID NO: 60, 566) 16. SERPINA1_T444GGAGAGGATGTGCAGGGCCA (SEQ ID NO: 60, 567) 32.7 SERPINA1_T113TCACTCCCCCTGGACGGCCC (SEQ ID NO: 55, 728) 9.7 SERPINA1_T253GTGCAGGGCCAGGGCCGTCC (SEQ ID NO: 60, 569) 67.9 SERPINA1_T312TGCAGGGCCAGGGCCGTCCA (SEQ ID NO: 60, 570) 63.7 SERPINA1_T445GCAGGGCCAGGGCCGTCCAG (SEQ ID NO: 60, 571) 59.7 SERPINA1_T441CAGGGCCAGGGCCGTCCAGG (SEQ ID NO: 60, 572) 55.8 SERPINA1_T51AAGCGCTCACTCCCCCTGGA (SEQ ID NO: 55, 727) 72.9 SERPINA1_T114CAGGAAGCGCTCACTCCCCC (SEQ ID NO: 55, 726) 69.2 SERPINA1_T164CAGGGGGAGTGAGCGCTTCC (SEQ ID NO: 60, 575) 56.2 SERPINA1_T155AGGGGGAGTGAGCGCTTCCT (SEQ ID NO: 60, 576) 37.3 SERPINA1_T240GGGAGTGAGCGCTTCCTGGG (SEQ ID NO: 60, 578) 71.3 SERPINA1_T46GGCTCACGTGGACACCTCCC (SEQ ID NO: 55, 724) 65.4 SERPINA1_T30GGCCTCGAGCAAGGCTCACG (SEQ ID NO: 55, 722) 72.2 SERPINA3_T7GTCCACGTGAGCCTTGCTCG (SEQ ID NO: 60, 581) 58.6 SERPINA1_T209CTGATCCCAGGCCTCGAGCA (SEQ ID NO: 55, 721) 69.2 SERPINA1_T476CGTGAGCCTTGCTCGAGGCC (SEQ ID NO: 60, 582) 13.4 SERPINA1_T67GTGAGCCTTGCTCGAGGCCT (SEQ ID NO: 60, 583) 0.2 SERPINA1_T34CACGTTGTAAGGCTGATCCC (SEQ ID NO: 55, 718) 66 SERPINA3_T139AGAAGCAGAGACACGTTGTA (SEQ ID NO: 55, 716) 76.4 SERPINA1_T49GCCTTATGCACGGCCTGGAG (SEQ ID NO: 55, 709) 66.2 SERPINA1_T19AGCCTTATGCACGGCCTGGA (SEQ ID NO: 55, 708) 74.2 SERPINA1 T52CAGCCTTATGCACGGCCTGG (SEQ ID NO: 55, 707) 25.1 SERPINA1_T32GCACAGCCTTATGCACGGCC (SEQ ID NO: 55, 705) 65.9 SERPINA1_T54TCCCCTCCAGGCCGTGCATA (SEQ ID NO: 60, 588) 73.8 SERPINA3_T107GGTCAGCACAGCCTTATGCA (SEQ ID NO: 55, 704) 82.2 SERPINA1_T9TTCAGTCCCTTTCTCGTCGA (SEQ ID NO: 55, 701) 42 SERPINA1_T5GTGCTGACCATCGACGAGAA (SEQ ID NO: 60, 591) 53.9 SERPINA1_T14TGCTGACCATCGACGAGAAA (SEQ ID NO: 60, 592) 40.5 SERPINA1_T355GAGAAAGGGACTGAAGCTGC (SEQ ID NO: 60, 594) 15.3 SERPINA3_T75GGGTATGGCCTCTAAAAACA (SEQ ID NO: 55, 697) 33.5 SERPINA1_T175TGCTGGGGCCATGTTTTTAG (SEQ ID NO: 60, 599) 45.3 SERPINA1_T213GGGGGGGATAGACATGGGTA (SEQ ID NO: 55, 696) 55 SERPINA1_T11ACCTCGGGGGGGATAGACAT (SEQ ID NO: 55, 695) 39.3 SERPINA1_733GACCTCGGGGGGGATAGACA (SEQ ID NO: 55, 694) 49.4 SERPINA1_T6ACCCATGTCTATCCCCCCCG (SEQ ID NO: 60, 601) 66.5 SERPINA1_T31TGTTGAACTTGACCTCGGGG (SEQ ID NO: 55, 692) 13.3 SERPINA1_T29TTGTTGAACTTGACCTCGGG (SEQ ID NO: 55, 691) 4.6 SERPINA1_T23TTTGTTGAACTTGACCTCGG (SEQ ID NO: 55, 690) 36.4 SERPINA1_T18GTTTGTTGAACTTGACCTCG (SEQ ID NO: 55, 689) 20 SERPINA1_T74GGTTTGTTGAACTTGACCTC (SEQ ID NO: 55, 688) 56.9 SERPINA1_T63GGGTTTGTTGAACTTGACCT (SEQ ID NO: 55, 687) 32.2 SERPINA1_T317TCAATCATTAAGAAGACAAA (SEQ ID NO: 55, 686) 63.2 SERPINA1_T276TTCAATCATTAAGAAGACAA (SEQ ID NO: 55, 685) 15.5 SERPINA1_T365TCCCATGAAGAGGGGAGACT (SEQ ID NO: 55, 681) 28.2 SERPINA1_T188TACCAAGTCTCCCCTCTTCA (SEQ ID NO: 60, 604) 27.4 SERPINA1_T243ACCAAGTCTCCCCTCTTCAT (SEQ ID NO: 60, 605) 46.2 SERPINA1_T289CACCACTTTTCCCATGAAGA (SEQ ID NO: 55, 678) 20.6 SERPINA1_T293TCACCACTTTTCCCATGAAG (SEQ ID NO: 55, 677) 24.5 SERPINA1_T334TCCCCTCTTCATGGGAAAAG (SEQ ID NO: 60, 607) 48.9 SERPINA1_T252AGAGGCAGTTATTTTTGGGT (SEQ ID NO: 55, 674) 14.1 SERPINA1_T346GAGAGGCAGTTATTTTTGGG (SEQ ID NO: 55, 673) 9.5 SERPINA1_T134AGCGAGAGGCAGTTATTTTT (SEQ ID NO: 55, 672) 47.8 SERPINA1_T331GAGCGAGAGGCAGTTATTTT (SEQ ID NO: 55, 671) 54.3 SERPINA1_T399GGGAGGGGTTGAGGAGCGAG (SEQ ID NO: 55, 669) 54.1 SERPINA1_T464GGGAGGGGGCCAGGGATGGA (SEQ ID NO: 55, 658) 13.9 SERPINA1_T460AGGGAGGGGGCCAGGGATGG (SEQ ID NO: 55, 657) 1.5 SERPINA1_T462CAACCCCTCCCCTCCATCCC (SEQ ID NO: 60, 608) 21.9 SERPINA1_T256CCCTTCTTTAATGTCATCCA (SEQ ID NO: 55, 646) 55 5E5PINA1_T96ACCCTTCTTTAATGTCATCC (SEQ ID NO: 55, 645) 35.6 SERPINA1_T229ACATTAAAGAAGGGTTGAGC (SEQ ID NO: 60, 615) 30 SERPINA1_T361TTTACAGTCACATGCAGGCA (SEQ ID NO: 55, 642) 50.7 SERPINA1_T201ATTTACAGTCACATGCAGGC (SEQ ID NO: 55, 641) 49.4 SERPINA1_T132AGGGATTTACAGTCACATGC (SEQ ID NO: 55, 639) 70.7 SERPINA1_T316AGACTCAGAGAAAACATGGG (SEQ ID NO: 55, 635) 43.6 SERPINA1_T302AGACTCAGAGAAAACATGGG (SEQ ID NO: 55, 633) 41.3 SERPINA1_T271ATACAGCCTCAGCAGGCAAA (SEQ ID NO: 55, 628) 67.7 SERPINA1_T193CATACAGCCTCAGCAGGCAA (SEQ ID NO: 55, 627) 40.5 SERPINA1_T340GAGTCTCCCTTTGCCTGCTG (SEQ ID NO: 60, 618) 72.6 SERPINA1_T169AGCCCACATACAGCCTCAGC (SEQ ID NO: 55, 625) 49.8 5E5PINA1_T129TTGCCTGCTGAGGCTGTATG (SEQ ID NO: 60, 619) 74.3 SERPINA1_T145TGCCTGCTGAGGCTGTATGT (SEQ ID NO: 60, 620) 41.2 SERPINA1_T204TGAGGCTGTATGTGGGCTCC (SEQ ID NO: 60, 622) 42.6 SERPINA1_T35CCGAAGACAGCACTGTTACC (SEQ ID NO: 55, 620) 29 SERPINA1_T184CCAGGTAACAGTGCTGTCTT (SEQ ID NO: 60, 624) 0 SERPINA1_T178CAGGTAACAGTGCTGTCTTC (SEQ ID NO: 60, 625) 0 SERPINA1_T157CTCCATGAACACAGTTCAGG (SEQ ID NO: 55, 617) 0 SERPINA1_T140GCTCCATGAACACAGTTCAG (SEQ ID NO: 55, 616) 41.6 SERPINA1_T147AACTGTGTTCATGGAGCATC (SEQ ID NO: 60, 628) 18.2 SE5PINA1_T273TGTTCATGGAGCATCTGGCT (SEQ ID NO: 60, 630) 61.9 SERPINA1_T247CATGGAGCATCTGGCTGGGT (SEQ ID NO: 60, 632) 49.1 SERPINA1_T380CTGGCTGGGTAGGCACATGC (SEQ ID NO: 60, 633) 77.7 SERPINA1_T456CACATGCTGGGCTTGAATCC (SEQ ID NO: 60, 636) 0 SERPINA1_T352ACATGCTGGGCTTGAATCCA (SEQ ID NO: 60, 637) 63.2 SE5PINA1_T443ATGCTGGGCTTGAATCCAGG (SEQ ID NO: 60, 639) 71.8 SERPINA1_T377TGCTGGGCTTGAATCCAGGG (SEQ ID NO: 60, 640) 57.3 SERPINA1_T76GCTGAGGATTCAGTCCCCCC (SEQ ID NO: 55, 607) 80.9 SERPINA1_T44GGGCCCAGGTCCGTAAGGTG (SEQ ID NO: 55, 605) 42.7 SERPINA1_T160GGGACTGAATCCTCAGCTTA (SEQ ID NO: 60, 642) 58.7 SERPINA1_T4GAATCCTCAGCTTACGGACC (SEQ ID NO: 60, 643) 13.7 SERPINA1_T20AATCCTCAGCTTACGGACCT (SEQ ID NO: 60, 644) 6.1 SERPINA1_T207CTCCAGAAACAGATGGGCCC (SEQ ID NO: 55, 602) 43 SERPINA1_T260GGAGCCCTCCAGAAACAGAT (SEQ ID NO: 55, 600) 74.5 SERPINA1_T403TGGAGCCCTCCAGAAACAGA (SEQ ID NO: 55, 599) 37.8 SERPINA1_T87GACCTGGGCCCATCTGTTTC (SEQ ID NO: 60, 645) 49.4 SERPINA1_T284CTGGGCCCATCTGTTTCTGG (SEQ ID NO: 60, 647) 32.7 SERPINA1_T228TGGGCCCATCTGTTTCTGGA (SEQ ID NO: 60, 648) 72.4 SERPINA1_T406CAAGACAGGACAAGGAAGAC (SEO ID NO: 55, 595) 22.2 SERPINA1_T304CAGTCTTCCTTGTCCTGTCT (SEQ ID NO: 60, 650) 37.4 SERPINA1_T126CTTCTTGGGGACTCCAAGAC (SEQ ID NO: 55, 591) 30.1 SERPINA1_T137CTGTCTTGGAGTCCCCAAGA (SEQ ID NO: 60, 654) 0 SERPINA1_T324CTCCCCTGTGATTCCTTCTT (SEQ ID NO: 55, 587) 41.3 SERPINA1_T367CCTCCCCTGTGATTCCTTCT (SEQ ID NO: 55, 586) 46.3 SERPINA1_T248GTCCCCAAGAAGGAATCACA (SEQ ID NO: 60, 657) 51 SERPINA1_T310TCCCCAAGAAGGAATCACAG (SEQ ID NO: 60, 658) 27.8 SERPINA1_T342CCAAGAAGGAATCACAGGGG (SEQ ID NO: 60, 660) 49 SERPINA1_T332GTGGAGGCTGGGGTCATGGC (SEQ ID NO: 55, 584) 48.7 SERPINA1_T358CTTGGTGGAGCCTGGGGTCA (SEQ ID NO: 55, 583) 30 SERPINA1_T395CAGATACCAGCCATGACCCC (SEQ ID NO: 60, 664) 20.8 SERPINA1_T362AAGATGCTTGGTGGAGCCTG (SEQ ID NO: 55, 582) 21.1 SERPINA1_T235GAAGATGCTTGGTGGAGCCT (SEQ ID NO: 55, 581) 4.1 SERPINA1_T238TGAAGATGCTTGGTGGAGCC (SEQ ID NO: 55, 580) 5.8 SERPINA1_T255GGGGACATGAAGATGCTTGG (SEQ ID NO: 55, 578) 59.5 SERPINA1_T296CAGGGGGACATGAAGATGCT (SEQ ID NO: 55, 577) 64.9 SERPINA1_T455GGGAGTGGGGGATGAGCAGG (SEQ ID NO: 55, 575) 43.7 SERPINA1_T347GGGGAGTGGGGGATGAGCAG (SEQ ID NO: 55, 574) 67.5 SERPINA1_T387GGGGGAGTGGGGGATGAGCA (SEQ ID NO: 55, 573) 27 SERPINA1_T416GGGGGGAGTGGGGGATGAGC (SEQ ID NO: 55, 572) 15.8 SERPINA1_T440CTCTGGGTGGGGGGGAGTGG (SEQ ID NO: 55, 569) 31.4 SERPINA1_T430ACTCTGGGTGGGGGGGAGTG (SEQ ID NO: 55, 568) 11.8 SERPINA1_T389AACTCTGGGTGGGGGGGAGT (SEQ ID NO: 55, 567) 6.7 SERPINA1_T398CAACTCTGGGTGGGGGGGAG (SEQ ID NO: 55, 566) 6.1 SERPINA1_T415ATGAGCAACTCTGGGTGGGG (SEQ ID NO: 55, 564) 35.7 SERPINA1_T412GATGAGCAACTCTGGGTGGG (SEQ ID NO: 55, 563) 30.3 SERPINA1_T396GGATGAGCAACTCTGGGTGG (SEQ ID NO: 55, 562) 30.4 SERPINA1_T426AGGATGAGCAACTCTGGGTG (SEQ ID NO: 55, 561) 18.5 SERPINA1_T246CAGGATGAGCAACTCTGGGT (SEQ ID NO: 55, 560) 35.1 SERPINA1_T373GCAGGATGAGCAACTCTGGG (SEQ ID NO: 55, 559) 40.8 SERPINA1_T205CTGGCAGGATGAGCAACTCT (SEQ ID NO: 55, 558) #N/A SERPINA1_T174CCTGGCAGGATGAGCAACTC (SEQ ID NO: 55, 557) #N/A SERPINA1_T219CCAGAGTTGCTCATCCTGCC (SEQ ID NO: 60, 669) #N/A SERPINA1_T171CAGAGTTGCTCATCCTGCCA (SEQ ID NO: 60, 670) #N/A

Note Regarding Illustrative Embodiments

While the present disclosure provides descriptions of various specificembodiments for the purpose of illustrating various aspects of thepresent invention and/or its potential applications, it is understoodthat variations and modifications will occur to those skilled in theart. Accordingly, the invention or inventions described herein should beunderstood to be at least as broad as they are claimed, and not as morenarrowly defined by particular illustrative embodiments provided herein.

Any patent, publication, or other disclosure material identified hereinis incorporated by reference into this specification in its entiretyunless otherwise indicated, but only to the extent that the incorporatedmaterial does not conflict with existing descriptions, definitions,statements, or other disclosure material expressly set forth in thisspecification. As such, and to the extent necessary, the expressdisclosure as set forth in this specification supersedes any conflictingmaterial incorporated by reference. Any material, or portion thereof,that is said to be incorporated by reference into this specification,but which conflicts with existing definitions, statements, or otherdisclosure material set forth herein, is only incorporated to the extentthat no conflict arises between that incorporated material and theexisting disclosure material. Applicants reserve the right to amend thisspecification to expressly recite any subject matter, or portionthereof, incorporated by reference herein.

What is claimed is:
 1. One or more guide ribonucleic acids (gRNAs) forediting a SERPINA1 gene in a cell from a patient with alpha-1antitrypsin deficiency (AATD), the one or more gRNAs comprising a spacersequence selected from the group consisting of the RNA sequencescorresponding to the full-length nucleic acid sequences in SEQ ID NOs:55,559, 55,574, 55,575, 55,577, 55,578, 55,584, 55,586, 55,587, 55,600,55,602, 55,605, 55,607, 55,616, 55,625, 55,627, 55,628, 55,633, 55,635,55,639, 55,641, 55,642, 55,646, 55,669, 55,671, 55,672, 55,686, 55,688,55,694, 55,696, 55,701, 55,704, 55,705, 55,708, 55,709, 55,716, 55,718,55,721, 55,722, 55,724, 55,726, 55,727, 55,730, 55,732, 55,743, 55,744,55,748, 55,749, 55,751, 55,754, 55,762, 55,764, 55,771, 55,778,55,781-55,783, 60,476, 60,477, 60,480-60,482, 60,485, 60,487, 60,490,60,494, 60,496, 60,497, 60,504, 60,506, 60,515, 60,517, 60,519, 60,527,60,529, 60,539-60,541, 60,555, 60,557, 60,558, 60,564, 60,569-60,572,60,575, 60,578, 60,581, 60,588, 60,591, 60,592, 60,599, 60,601, 60,605,60,607, 60,618-60,620, 60,622, 60,630, 60,632, 60,633, 60,637, 60,639,60,640, 60,642, 60,645, 60,648, 60,657, and 60,660, wherein said one ormore gRNAs has a cutting efficiency of greater than 40%.
 2. The one ormore gRNAs of claim 1, wherein the one or more gRNAs are one or moresingle-molecule guide RNAs (sgRNAs).
 3. The one or more gRNAs of claim1, wherein the one or more gRNAs is one or more modified gRNAs.
 4. A kitfor editing a SERPINA1 gene in a human cell, the kit comprising: (a) apolynucleotide donor template comprising a nucleic acid sequenceencoding at least a portion of the wild-type SERPINA1 gene; (b) at leastone polypeptide comprising the amino acid sequence of a site-directedendonuclease or at least one polynucleotide comprising a nucleic acidsequence encoding said site-directed endonuclease; and (c) one or moreguide ribonucleic acids (gRNAs) comprising a spacer sequence selectedfrom the group consisting of the RNA sequences corresponding to thefull-length nucleic acid sequences of SEQ ID NOs: 55,559, 55,574,55,575, 55,577, 55,578, 55,584, 55,586, 55,587, 55,600, 55,602, 55,605,55,607, 55,616, 55,625, 55,627, 55,628, 55,633, 55,635, 55,639, 55,641,55,642, 55,646, 55,669, 55,671, 55,672, 55,686, 55,688, 55,694, 55,696,55,701, 55,704, 55,705, 55,708, 55,709, 55,716, 55,718, 55,721, 55,722,55,724, 55,726, 55,727, 55,730, 55,732, 55,743, 55,744, 55,748, 55,749,55,751, 55,754, 55,762, 55,764, 55,771, 55,778, 55,781-55,783, 60,476,60,477, 60,480-60,482, 60,485, 60,487, 60,490, 60,494, 60,496, 60,497,60,504, 60,506, 60,515, 60,517, 60,519, 60,527, 60,529, 60,539-60,541,60,555, 60,557, 60,558, 60,564, 60,569-60,572, 60,575, 60,578, 60,581,60,588, 60,591, 60,592, 60,599, 60,601, 60,605, 60,607, 60,618-60,620,60,622, 60,630, 60,632, 60,633, 60,637, 60,639, 60,640, 60,642, 60,645,60,648, 60,657, and 60,660, wherein said one or more gRNAs has a cuttingefficiency of greater than 40%.
 5. The one or more sgRNAs of claim 2,wherein the one or more sgRNAs is one or more modified sgRNAs.